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Effect of elevated temperature and water availability on CO2 exchange and nitrogen fixation of nodulated alfalfa plants
Environmental and Experimental Botany 59 (2007) 99–108
Effect of elevated temperature and water availability on CO2 exchange and nitrogen fixation of nodulated alfalfa plants Iker Aranjuelo, Juan Jos´e Irigoyen, Manuel S´anchez-D´ıaz ∗ Departamento de Fisiolog´ıa Vegetal, Facultades de Ciencias y Farmacia, Universidad de Navarra, Irunlarrea s/n, 31008 Pamplona, Spain Received 21 December 2004; received in revised form 7 June 2005; accepted 31 October 2005
Abstract The aim of this study was to investigate the effects of predicted temperature increases and drought conditions of Mediterranean environments on N2 -fixing alfalfa plants. One-month-old plants inoculated with Sinorhizobium meliloti strain 102F78 were grown in growth chambers under different temperature (25/15 or 28/18 ◦ C, day/night) and water availability (control or drought) regimes. Elevated temperature and drought reduced plant dry mass and leaf area, especially when both stresses were combined. The inhibitory effect of elevated temperature on plant growth was a consequence of decreased CO2 and N2 fixation rates. A photosynthetic decrease resulted from the inhibition of rubisco activity, probably associated with a lower activation state. An absence of differences in photosynthesis in relation to water availability suggests that drought decreased plant growth due to its negative effect on leaf area. Rising temperature and drought affected the nitrogen content negatively, although effects differed. Elevated temperature inhibited nodule activity drastically, whereas the inhibitory effect resulting from drought centred on nodule dry mass (DM) production. Plants exposed to a combination of elevated temperature and drought were the most negatively affected. © 2005 Elsevier B.V. All rights reserved. Keywords: Alfalfa; Drought; Nitrogen; Nodule; Photosynthesis; Temperature
1. Introduction General circulation models (GCM) predict that ambient temperature in the Mediterranean basin is going to increase 1–3 ◦ C by the middle of this century. Rising temperature will increase evapotranspiration rates and exacerbate low water availability problems commonly observed in Mediterranean environments, in which current annual potential evapotranspiration is often nearly twice the amount of rainfall (S´abate et al., 2002). It has long been known that drought during the warmest period of the year is the major stress factor limiting plant species distribution and growth in Mediterranean regions of the world (Mooney, 1983). Most studies analysing the effect of Mediterranean environments on plant growth have considered temperature and water availability separately. However, it is well known that the effect of combined stresses on plant growth causes alterations that cannot ∗
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0098-8472/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2005.10.008
be predicted if they are analysed alone, such as those resulting from synergistic and antagonistic phenomena (Valladares and Pearcy, 1997). For example, Chaves and Pereira (2004) observed that although photochemical processes are very resistant to low water availability, a down regulation of the photosynthetic apparatus occurs when plants are exposed simultaneously to drought and elevated temperature conditions. In addition, we would like to highlight the observation of Kaiser (1987) suggesting that the particular way in which water stress is imposed might be of special importance not only in understanding the field response to drought, but also in evaluating the plant’s capacity to acclimate to stress. Withholding water is the most common method for short-term experiments, but sustained or cyclic water stress is needed to simulate more realistic responses to drought (Pennypacker et al., 1990). The inhibitory effects of elevated temperatures target photosynthesis, water status, and N2 fixation in the case of N2 -fixing plants (Bushby, 1982). Many publications show
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that the photosynthetic rates of evergreen species in Mediterranean environments during summer drought decrease due to stomatal closure (Pe˜nuelas et al., 1998; Llusi`a and Pe˜nuelas, 2000) and metabolic limitation (rubisco activity, RuBP regeneration, etc.) (Tretiach et al., 1997; Larcher, 2000). CO2 fixation may be affected by an altered excitation energy distribution associated with changes of thylakoid membrane fluidity and by damaged Calvin cycle enzyme activities. Enhanced CO2 /O2 solubility coupled with diminished specificity of rubisco for CO2 associated with plants grown under high temperature is reflected in enhanced photorespiration ¨ and decreased photosynthesis rates (Jordan and Ogren, 1984; Brooks and Farquhar, 1985). Besides, Stitt et al. (1991) explained reduced CO2 fixation rates as a consequence of lower photoassimilate requirements, which lead to carbohydrate accumulation and the inhibition of Calvin cycle enzyme activities. The sensitivity of legume-Sinorhizobium symbiosis to ambient stresses like temperature and low water availability is also well known (Antol´ın and S´anchez-D´ıaz, 1992; Serraj et al., 1999; Hungria and Vargas, 2000; Streeter, 2003; Curtis et al., 2004; Thomas et al., 2004). Furthermore, some authors (Castellanos et al., 1996; Thomas et al., 2004) suggest that the effect of water deficit on N accumulation and N2 fixation is larger than that on biomass accumulation. Alfalfa is a temperate forage frequently exposed to low water availability, N-deficient soils and high temperature conditions. Photosynthesis supplies organic carbon to nodules, where it is used by the nitrogenase enzyme in the bacteroid inside nodules as a source of energy and reducing power to fix N2 (Azc´on-Bieto et al., 2000). The products of N2 fixation, either amides or ureids, are exported to the plant via the xylem (Schubert et al., 1995; Walsh, 1995). Factors that increase photosynthesis increase N2 fixation, while factors decreasing photosynthesis tend to decrease N2 fixation. This coupling results in the regulation of nitrogenase activity in plants by photosynthesis (carbon supply), nitrogen availability (N source strength) and N demand (N sink strength). In order to investigate the deleterious effect of elevated temperature and drought on CO2 fixation and its relation with N2 assimilation, we analysed rubisco activity, carbohydrate concentration, and several nodule enzymes involved in C and N metabolism. Hexoses provided by the plant are hydrolysed in the nodule to obtain phosphoenolpyruvate through the glycolytic pathway. Phosphoenolpyruvate carboxylase (PEPC) then catalyses the combination of respiratory CO2 (as HCO3 − ) with phosphoenolpyruvate to produce oxaloacetate, which is converted to malate by malate dehydrogenase (MDH). This malate can either be used as a source of carbon and energy for bacteroid consumption, enter mitochondria to be oxidised in the tricarboxylic acid cycle, or contribute to ammonia assimilation in the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle (Cabrerizo et al., 2001). MDH also forms a complex with the glutamate oxaloacetate transaminase (GOT) enzyme. This complex is responsible for transamination between glutamate and
oxaloacetate to yield 2-oxoglutarate and aspartate, which is converted into asparagine by asparagine synthetase. A dicarboxylic acid like malate, whilst excluding other putative substrates like ␣-ketoglutarate, glutamate, pyruvate and arabinose, is a likely candidate to support N2 fixation in nodules (Udvardi and Day, 1997). Serraj et al. (1999) suggest that N2 fixation is also regulated by nodule permeability to O2 and feedback regulation by N compounds under conditions of low water availability. A lower rate of water movement out of the nodule during drought stress may restrict export of N2 fixation products, thus inhibiting nitrogenase activity via a feedback mechanism (Pate et al., 1969; Serraj et al., 1999). The optimum temperature range for root-nodule symbiosis is between 25 and 33 ◦ C (Pankhurst and Sprent, 1976), although reductions in N2 fixation activity have been observed above 28 ◦ C (Hungria and Franco, 1993). Elevated temperature might affect N2 fixation directly or indirectly. Direct inhibition by temperature is a consequence of decreased nodule development (Dart and Mercer, 1965; Piha and Munns, 1987), functionality (Meyer and Anderson, 1959; Piha and Munns, 1987; Hern´andez-Armenta et al., 1989) and accelerated nodule senescence. Indirect inhibition is related to temperature effects on root hair formation depression, reduction of nodulation sites (Frings, 1976; Jones and Tisdale, 1921), and modified adherence of bacteria to root hairs (Frings, 1976). The main objective of the present study is to investigate the effect of predicted temperature and low water availability conditions on N2 -fixing alfalfa plant growth. In order to analyse the effect of elevated temperature and drought on nodulated alfalfa plants, experiments were performed in growth chambers in which plants were exposed to conditions differing in temperature (25 ◦ C versus 28 ◦ C) and water availability (control versus drought). It was predicted that temperature and drought would reduce plant growth as a consequence of their negative effects on water status, gas exchange, and N2 fixation. In order to test such a hypothesis, the effects of elevated temperature and water availability on water status, gas exchange and nodule metabolism were analysed. Plant water status was evaluated by the measurement of leaf relative water content (RWC). Stomatal (leaf conductance and intercellular CO2 concentration) and non-stomatal (light capture and rubisco carboxylation activity) measurements were carried out to determine the effects of elevated temperature and drought on CO2 fixation rates. Nodule N2 fixation was analysed through the analysis of several nodule plant and bacteroid key enzymes involved in nodule C and N metabolism.
2. Material and methods 2.1. Plant material and experimental design Seedlings of alfalfa (Medicago sativa L. cv. Arag´on) were transferred into 2 L pots (20 plants per pot) containing
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a mixture of inert perlite and vermiculite (2/1, v/v). During the first month, plants were inoculated three times with Sinorhizobium meliloti strain 102F78 (The Nitragin Co., Milwaukee, WI, USA) and grown in a greenhouse at 25/15 ◦ C (day/night) with a photoperiod of 14 h under natural daylight, supplemented with fluorescent lamps (Sylvania DECOR 183, Professional-58W, Germany) that provided a photosynthetic photon flux density (PPDF) of about 300–400 mol m−2 s−1 . Plants were watered twice a week with Evans N-free nutrient solution (Evans, 1974) and once a week with tap water to avoid salt accumulation in pots. When 30 days old, plants were transferred to controlled environment chambers (Conviron PGV 36, Winnipeg, Canada) and randomly assigned to four treatments (eight pots per treatment): well-watered and water-stressed plants grown at a temperature of 28 ◦ C (elevated) or 25 ◦ C (control). Plants were maintained in growth chambers under the specified growth conditions for a period of 30 days, by the end of which plants were 60 days old. Pots were rotated every week from one chamber to another to avoid chamber effects. Well-watered plants were irrigated until a maximum soil volumetric water content (θ v ) of around 400 mm3 mm−3 was reached. The drought level used in treatments corresponded to 70% θ v of well-watered plants (around 280 mm3 mm−3 ). Such θ v levels were maintained throughout the experiment by measuring the transpired water daily (calculated by weighing the pots) and replenishing the lost water. To reduce soil evaporation, pots were covered with plastic sheets perforated with very small holes to allow stems to pass through. The desired drought level was reached around 12 days after the beginning of the treatment, when plants were 42 days old. Fully irrigated plants were alternatively watered with Evans N-free nutrient solution (Evans, 1974) and distilled water, whereas plants subjected to drought were always watered with Evans solution in order to supply all treatments with the same amount of nutrients. Conditions in the growth chamber involved temperatures of 25/15 ◦ C (day/night, low temperature) and 45% RH (1.7 kPa vapor pressure deficit, VPD) or 28/18 ◦ C (day/night, elevated temperature) and 54% RH (1.7 kPa VPD), ambient CO2 of around 400 mol mol−1 , a 14 h photoperiod and 600 mol m−2 s−1 PPFD. The experiment was repeated in two consecutive years. Experiments in the second year were performed to confirm results obtained in the first year. Values presented in this paper correspond to mean values of data collected for both years. Temperature was determined at leaf and root levels. Measurements corresponding to root levels were obtained from the soil surface and at a depth of 5 cm (where most of the nodules develop). Soil temperature was measured with a thermocouple thermometer. Leaf temperature was measured daily on adaxial and abaxial surfaces of the youngest fully expanded leaves, where gas exchange and chlorophyll fluorescence were measured. Leaf temperature was determined using a non-contact infrared thermometer LP-H (Raytec, Rynger PK, Berlin, Germany). Measurements were performed under growth conditions inside the growth chamber. Apical, fully
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expanded leaves without symptoms of injury were selected for each determination. 2.2. Growth parameters and water relations To record plant growth parameters, the first harvest was performed when the plants reached the desired θ v level (42day-old plants), and the second harvest was performed on the last day of each treatment (60-day-old plants). Plant organs were weighed separately for both harvests after being separated into leaves, stems, roots and nodules. The dry mass (DM) of each organ was obtained after drying in an oven at 80 ◦ C for 48 h. A root:shoot ratio was calculated as the proportion of root and nodule DM versus leaf and stem DM. Leaf area was measured directly using an automatic leaf area meter (Li-3000, LiCor, NE, USA). Plant water status was evaluated by measuring the relative water content of a leaf (Weatherley, 1950). Soil volumetric water content was calculated by weighing pots every day at the beginning of the photoperiod. Water use efficiency of productivity (WUEp ) is calculated as the ratio of produced dry mass to the water consumption in a period of time (Larcher, 1995; De Luis et al., 1999). WUEp was calculated for the period between the second harvest (42-day-old plants) and the final harvest (60-day-old plants). 2.3. Leaf determinations Fully expanded apical leaves from 50-day-old plants were individually enclosed in a leaf chamber (1010-M, Waltz, Effeltrich, Germany), and the gas exchange rate was measured with a portable photosynthesis system (HCM-1000, Waltz) under growth conditions. Net photosynthesis (A) and leaf conductance (g) were calculated as described by von Caemmerer and Farquhar (1981). Leaf internal CO2 concentration (Ci ) was estimated from net photosynthesis and conductance measurements according to Farquhar and Sharkey (1982). The optimal quantum yield of photosystem II was measured using a photosynthesis yield analyser (MINI-PAM, Waltz). Measurements were taken simultaneously with gas exchange determinations. Measuring conditions were the same as those of growth conditions, but the leaves were first dark-adapted for 20 min before carrying out the measurements. Leaf total soluble proteins (TSP), total soluble sugars (TSS) and starch concentration were quantified by grinding and filtering 100 mg of fresh weight frozen leaf tissue in a cold mortar using an extraction buffer containing 50 mM Kphosphate (pH 7.5). Extract was filtered and centrifuged at 28,710 × g for 15 min at 4 ◦ C. The supernatant was used for TSP and TSS quantification, whereas starch was measured in the pellet as described by Jarvis and Walker (1993). TSP levels were measured using the protein dye-binding method of Bradford (1976), while TSS determinations followed the procedure of Yemm and Willis (1954).
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Pigment concentration was determined in each leaf used for gas exchange and chlorophyll fluorescence analyses. Extracts for pigment analysis were prepared by grinding and filtering 100 mg fresh weight of the last fully developed leaves in a cold mortar with 10 ml of ethanol (95%, v/v). The homogenate was centrifuged at 3165 × g for 10 min at 4 ◦ C. The supernatant was used for pigment analyses. An aliquot of 1 ml from this extract was taken, 4 ml of ethanol 95% were added, and the absorbance measured at 750, 665, 649 and 470 nm. Absorbance determinations were carried out with a Spectronic 2000 (Bausch and Lomb, Rochester, USA) spectrophotometer. Extinction coefficients and equations used to calculate pigment contents were those described by Liechenthaler (1987). Rubisco (E.C. 4.1.1.39) extractable activity was determined in the same leaves used for gas exchange and chlorophyll fluorescence measurements. Enzyme activity was monitored by grinding and filtering 250 mg fresh weight of frozen leaf material, representing the last fully developed leaves, in a cold mortar using an extraction buffer containing 50 mM K-phosphate buffer (pH 7.5), 1 mM ethylenediaminetetraacetic acid iron(III) sodium salt (EDTA, Sigma–Aldrich), 8 mM MgCl2 ·6H2 O, 5 mM dl-dithiothreitol (freshly prepared, Sigma–Aldrich) and 1% (p/v) polyvinylpolypyrrolidone (PVPP, Sigma–Aldrich). The extract was then clarified by centrifugation at 26,890 × g for 10 min at 4 ◦ C. Enzyme activity in the extract was determined by measuring the absorbance at 340 nm, as described by Lilley and Walker (1974). 2.4. Nitrogen content and specific nodule activity Leaf, stem and root samples, previously dried at 60 ◦ C for 48 h, were ground in a mill with titanium blades and stored in vials placed in desiccators over silica gel. The nitrogen concentration was determined by means of sulphuric acid digestion in a B¨uchi K-424 (B¨uchi, Switzerland). Samples of 100 mg were digested by adding 20 ml H2 SO4 and a Kjeldhal Cu-Se catalytic pill. The digestion process ran for 1 h at 250 ◦ C until the sample was clarified. The samples were then diluted to 50 ml with distilled water. For the determination of nitrogen content, 5 ml of ionic strength adjuster (ISA, Ref. 951211, Orion, NY, USA) were added to 5 ml of measuring solution. Measurements were performed with an ammonia selective electrode (Orion 95-12BN, NY, USA) using 0.1 mM ammonium chloride as a standard. Specific nodule activity (SNA) was calculated as described by Brioua and Wheeler (1994), being the ratio between plant total nitrogen content and nodule DM. 2.5. Nodule determinations Five hundred milligrams of freshly harvested nodules were crushed in 10 ml of 50 mM K-phosphate buffer (pH 7.8), with 0.2% (v/v) 2-mercaptoethanol, 0.1 mM Na2 -EDTA and 10% (w/w) PVPP in a cold mortar. Isolation of plant and bac-
teroid fractions were performed as described by Irigoyen et al. (1992). To ensure no contamination between plant and bacterial fractions, phosphoenolpyruvate carboxylase was used as a plant fraction enzyme marker. No PEPC activity was detected for the bacteroid fraction. Phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) activity was assayed in nodule plant fractions by monitoring NADH oxidation spectrophotometrically. The reaction was performed as described by Deroche et al. (1983). Malate dehydrogenase (E.C. 1.1.1.37) and isocitrate dehydrogenase (ICDH, E.C. 1.1.1.42) enzymes were analysed in both plant and bacteroid fractions. The reaction medium and assay conditions used are detailed in Vance and Stade (1984). Malate dehydrogenase was measured by monitoring NADH oxidation at 340 nm, whereas ICDH was determined by recording the NADPH change at 340 nm. Glutamate oxaloacetate transaminase (E.C.2.6.1.1) was assayed only in the plant fraction, according to the method detailed in Bergmeyer (1974). Total soluble protein was determined in both plant and bacteroid fractions, whereas TSS was only determined in plant fractions. Total soluble protein and TSS determination on aliquots of nodule extraction were performed as described in the leaf determination section. 2.6. Statistical analysis Two factor analyses of variance (ANOVA, Sokal and Rohlf, 1986) at 0.5 and 0.1% levels were performed to partition the variance into main effects and an interaction between the two factors (temperature and drought). When the F-ratio was significant, least significant differences were evaluated using the Tukey b-test (P < 0.05).
3. Results The leaf temperature of plants grown at 28 ◦ C was higher than that of plants grown at 25 ◦ C for both irrigation treatments (Fig. 1). Drought did not alter leaf temperature (Fig. 1). The same response was observed at the substrate surface level and at a depth of 5 cm (Fig. 1). Total dry mass was negatively affected by elevated temperature (28 ◦ C) in both fully and partially watered treatments (Table 1). Drought also reduced plant production. Plants exposed to both elevated temperature and drought incurred the greatest decrease in plant production. Individual DM of leaves, shoots and roots was negatively affected by the combination of both stress factors. Leaf area was negatively affected by low water availability, whereas only temperature decreased leaf area in partially watered treatments. Elevated temperature did not change the root:shoot ratio, which increased in response to low water availability. Leaf relative water content was not affected by temperature, whereas drought conditions had a negative effect on leaf RWC (Table 1). Analysis of soil volumetric water content (Table 1) revealed that fully watered plants had a higher soil
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Table 2 The effect of elevated temperature and drought on net photosynthesis (A), rubisco extractable activity, leaf total soluble sugars (TSS) and starch concentration of nodulated alfalfa plants (mol m−2 s−1 )
A Rubisco (mol g−1 DM min−1 ) TSS (mg g−1 DM) Starch (mg g−1 DM)
C25
C28
D25
D28
13.2a 20.0a 80.0ab 94.7b
8.4b 10.9b 53.1c 131.4a
14.1a 18.8a 86.0a 80.7b
10.5b 12.2b 64.6bc 99.9b
Each row represents the mean of 12 plants. Other details as in Table 1.
Fig. 1. The effect of elevated temperature and drought on leaf (adaxial) and soil (surface and 5 cm depth) temperature of nodulated alfalfa plants. Unshaded bars correspond to fully watered treatments; shaded bars correspond to partially watered plants. Each value represents the mean of 16 plants. Means with identical letters are not significantly different (P > 0.05).
water availability (≈30% more) when compared with partially watered plants, irrespective of temperature treatment. Although water use efficiency of productivity values were not changed significantly by temperature or water availability (Table 1), further analyses revealed that plants grown at 28 ◦ C produced less DM with lower water requirements. Plants grown at 28 ◦ C under fully and partially watered conditions suffered a reduction in net photosynthetic rate
(Table 2). CO2 fixation was not affected by water availability. There was no significant effect of temperature and drought on leaf conductance or intercellular CO2 concentration, and grand mean values were 108.2 mmol m−2 s−1 and 207.2 mol mol−1 , respectively. Rubisco extractable activity results were similar (Table 2) to observed photosynthesis measurements. Enzyme total activity was inhibited by elevated temperature, whereas no effect due to water availability was detected. It should be highlighted that there was no significant effect by temperature or water availability on total leaf soluble protein concentration (grand mean value of 70.3 mg g−1 DM). Elevated temperature decreased TSS (Table 2). No significant differences in starch concentration were detected between treatments, except for increased values recorded in fully watered plants grown under elevated temperature (Table 2). Leaf photosynthetic pigment content analyses showed that there was no appreciable change induced by temperature and/or water availability. Grand mean values for ChlA , ChlB , ChlA+B and Cx+c were 9.2, 2.9, 12.1 and 2.3 mg g−1 DM, respectively. There were no significant differences of Fv /Fm between treatments, which displayed a grand mean value of 0.8. Plant nitrogen content was inhibited by both temperature and water availability (Fig. 2). The interaction between elevated temperature and drought resulted in a 75% reduction of the N content. Specific nodule activity was significantly
Table 1 The effect of elevated temperature and drought on total dry mass (DM), leaf DM, stem DM, root DM, nodule DM, leaf area and root:shoot ratio, leaf relative water content (RWC), volumetric soil water content (θ v ), water use efficiency of productivity (WUEp ) and its components, DM production and water consumption of nodulated alfalfa plants Total DM (g plant−1 ) Leaf DM (g plant−1 ) Stem DM (g plant−1 ) Root DM (g plant−1 ) Nodule DM (mg plant−1 ) Leaf area (cm2 ) Root:shoot (g g−1 ) RWC (%) θ v (mm3 mm−3 ) WUEp (mg DM g−1 H2 O) DM production (g DM plant−1 ) Water consumption (g H2 O plant−1 )
C25
C28
D25
D28
2.2a 0.5a 0.7a 0.9a 39a 91.2a 0.8b 87.2a 480a 2.8a 2.0a 710.9a
1.3b 0.3b 0.4b 0.5b 43a 66.3ab 0.7b 84.7a 470a 2.6a 1.2b 451.0b
1.3b 0.3b 0.3b 0.7a 23b 43.9b 1.4a 76.0b 340b 3.1a 1.1b 357.3c
0.5c 0.1c 0.1c 0.2c 16b 26.9c 1.2a 76.2b 310b 2.1a 0.3c 165.9d
C25 and C28: fully watered plants grown under control (25 ◦ C) and elevated (28 ◦ C) temperature; D25 and D28: partially watered plants grown under control (25 ◦ C) and elevated (28 ◦ C) temperature. Each row represents the mean of 40 plants, except for RWC, which represents the mean of 12 plants. Means with identical letters (a–d) are not significantly different (P > 0.05).
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Fig. 2. The effect of elevated temperature and drought on plant total nitrogen content and specific nodule activity (SNA) of nodulated alfalfa plants. Each column represents the mean of 12 samples. Other details as in Fig. 1.
lower in elevated temperature treatments (28 ◦ C), whereas no significant effect was observed under conditions of low water availability (Fig. 2). Nodule plant fraction enzyme analyses showed that there were no differences on MDH activity between treatments, except for enhanced activity in fully watered plants grown under elevated temperature. An analysis of the separate effect of either temperature or water availability on GOT activity revealed that under optimal water availability conditions elevated temperature enhanced GOT activity whereas under low water availability conditions elevated temperature had inhibitory effect. The interaction of both stress factors reduced GOT activity significantly. Drought and elevated temperature alone did not affect PEPC activity, but PEPC enzyme activity decreased in plants exposed to both stress factors. An analysis of bacteroid fraction enzyme activity revealed that elevated temperature and drought inhibited MDH activity, especially when both parameters were combined (Table 3). There were no differences in ICDH activity between treatments. Nodule plant and bacteroid protein (Fig. 3) determinations showed that TSP content was neg-
Fig. 3. The effect of elevated temperature and drought on nodule bacteroid and plant total soluble protein (TSP) concentration of nodulated alfalfa plants. Each column represents the mean of 12 samples. Other details as in Fig. 1.
atively affected by elevated temperature in both nodule fractions. Low water availability decreased only the TSP in plant fractions. Total soluble sugars determinations did not reveal differences between treatments, and the grand mean value was 50.9 mg g−1 DM.
4. Discussion 4.1. Growth parameters and water relations Both elevated temperature and drought may limit plant growth (Aguirreolea and S´anchez-D´ıaz, 1989; Antol´ın and S´anchez-D´ıaz, 1992). Our study demonstrated that elevated temperature had a significant negative impact on biomass production, particularly when combined with drought. Increased temperature and drought reduced plant growth in a similar way (53 and 52%, respectively). Plants exposed to 28 ◦ C and drought were the most negatively affected (64% DM reduction). When analysing the effect of both stress factors on the different plant organs separately, a result similar
Table 3 The effect of elevated temperature and drought on nodule plant fraction malate dehydrogenase (MDH), glutamate oxaloacetate transaminase (GOT), phosphoenolpyruvate carboxylase (PEPC), isocitrate dehydrogenase (ICDH) and bacteroid fraction MDH and ICDH activities of nodulated alfalfa MDH (mmol g−1 DM min−1 ) GOT (mol g−1 DM min−1 ) PEPC (mol g−1 DM min−1 ) ICDH (mol g−1 DM min−1 ) MDH (mol g−1 DM min−1 ) ICDH (mol g−1 DM min−1 )
C25
C28
D25
D28
0.6b 124b 34a 87a 41a 1.5a
1.6a 150a 33a 19b 22b 1.0a
0.9b 129b 32a 17b 30b 1.7a
0.8b 96c 20b 13b 18c 1.0a
Each row represents the mean of 12 plants. Other details as in Table 1.
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to that of total DM is observed: elevated temperature and drought reduced leaf, shoot and root formation. Nodule dry mass production was not affected by increased temperature; however, low water availability had an inhibitory effect. Analyses of the root:shoot ratio revealed that the temperature increase affected above and belowground systems in a similar way. Plants subjected to drought had a larger root:shoot ratio, resulting mainly from a larger root biomass. Several authors (Hilbert and Canadell, 1995; Lloret et al., 1999) have hypothesized that this response may be an adaptation to the Mediterranean dry summer season. Variations in dry mass production observed between plant organs at elevated temperature could be explained by differences in sensitivity to high temperature (Sutcliffe, 1980). As shown with DM, leaf area was sensitive to increased temperature and drought. The absence of differences in CO2 fixation rates between fully and partially watered plants suggests that the inhibitory effect of low water availability on plant growth was centred on the leaf surface. Drought conditions and reduced leaf surface area resulted in decreased photosynthetic rates at the whole plant level. Diminished leaf area in plants subjected to drought could be the result of reduced cell division and elongation growth (Legg et al., 1975; Boyer et al., 1985), two processes extremely dependent on water availability. Leaf relative water content measurements revealed that differences observed in plant growth were due to temperature alone and not to an unequal water status, as the soil and leaf water contents were similar. According to Craufurd et al. (1999), heat stress might reduce WUE through the deleterious effect of temperature on carboxylation and respiration processes, and through alterations on water consumption. Although WUEp was not modified by temperature and water availability, analyses of WUEp components showed that they were affected negatively by both stress factors. Decreased water consumption of plants with the same leaf conductance is explained by their lower leaf surface, particularly in partially watered plants grown at 28 ◦ C. The absence of differences in WUEp could be explained by the fact that temperature and drought decreased DM production and water consumption simultaneously. 4.2. Leaf determinations As mentioned previously, the negative effects of elevated temperature and drought on alfalfa growth can be linked to changes in photosynthetic rates. Net photosynthesis can be diminished by stomatal limitations (associated with stomatal opening and closure) or non-stomatal limitations, generally corresponding to inhibitions of the photosynthetic apparatus (Escalona et al., 1999). Leaf conductance and intercellular CO2 concentration were unaffected by increased temperature or drought, which means that photosynthetic inhibition in plants grown at 28 ◦ C did not result from stomatal limitations. Although some authors report that stomata close at higher temperature, the closure probably results from a stomatal response to VPD that normally occurs when the
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leaf temperature is increased. As Berry and Bj¨orkman (1980) observed, when the same VPD is maintained, as was the case in this experiment, the stomata of plants exposed to elevated temperature remain open. Berry and Bj¨orkman (1980) also noted that stomata of plants grown under elevated temperature remained open at temperatures that cause damage to the photosynthetic apparatus. Decreased carboxylation efficiency might be a consequence of damage to photochemical reactions (Fellers et al., 1998). There may be a higher probability for photo-damage under limiting growth conditions (Baroli and Melis, 1998). The results of this study show that the optimum quantum yield of photosystem II, chlorophyll and carotenoid concentrations were within normal ranges, and that inhibition of photosynthesis was not caused by photochemical damage. These results are consistent with those of other studies (Oliveira and Pe˜nuelas, 2001; Ogaya and Pe˜nuelas, 2003) conducted in the Mediterranean basin, which showed that photoinhibition of PSII under drought conditions was only detected during the cold winter, and that no effect was detected in summer. Leegood and Edwards (1996) suggest that the mechanisms for reduced photosynthetic metabolism in C3 plants in response to increased temperature involve the enhancement of photorespiration and the inactivation of rubisco. The absence of differences in leaf respiration (data not shown) suggests that the decreased photosynthetic efficiency was a consequence of altered rubisco activity, as confirmed by rubisco extractable activity measurements. This reduction could be caused by the inactivation of the enzyme or by a reduction of enzyme concentration. The absence of differences in leaf total soluble protein suggests that decreased rubisco activity could be due to enzyme inactivation. According to several authors (Weis, 1981; Fellers et al., 1998), rubisco activity is regulated by rubisco activase. Some authors (Fellers et al., 1998; Escalona et al., 1999) have found that rubisco activase activity is sensitive to elevated temperature. Increased temperature may have inhibited rubisco activase, which would have reduced rubisco activity. It is also noteworthy that photosynthesis was not affected by water availability. In accordance with results obtained by other authors (De Luis, 2000; Ogaya and Pe˜nuelas, 2003), drought treatments tended to present net photosynthetic rates per soil water content that were higher than those of control treatments. This result indicates a quick and incipient acclimation to continuous low soil water availability, and suggests that plant growth is more sensitive than photosynthesis to low water availability regimes (Kramer and Boyer, 1995). 4.3. Nitrogen content and specific nodule activity As mentioned in the introduction, N2 -fixing plants such as legumes might also experience decreased plant growth due to the inhibitory effects of elevated temperature and drought on N2 fixation. The observed reduction in total nitrogen content in plants exposed to elevated temperature and low water availability can be explained by a decreased nodule dry mass
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formation and/or by diminished nodule activity. Specific nodule activity was analysed in an effort to determine which of these factors limited nitrogen fixation. The absence of an effect by drought on SNA indicates that low water availability inhibited N2 fixation through its deleterious effect on nodule DM production. These results accord with those obtained from Serraj and Sinclair (1998) through experiments performed in greenhouse and field conditions. Furthermore, the analysis of SNA also revealed that the decreased N content observed in plants grown at 28 ◦ C was caused by the negative effect of elevated temperature on nodule activity. 4.4. Nodule determinations To confirm previous observations, analyses were performed in plant and bacteroid fractions of several key nodule enzymes involved in nodule C and N metabolism, together with TSP, TSS and starch content. A general overview of nodule plant fraction metabolism results shows that drought did not modify enzyme activity. Furthermore, the plant fraction was more sensitive to elevated temperature, particularly under conditions of low water availability. Plants exposed to elevated temperature and drought had lower PEPC and GOT activities. PEPC provides up to 25% of the carbon required for nitrogenase activity and N assimilation (King et al., 1986). Reduction of PEPC suggests that the nodules of plants grown under conditions of elevated temperature and low water availability had an inadequate C supply. Furthermore, reduced PEPC activity also implies that less oxaloacetate was formed, and therefore the transamination between glutamate and oxaloacetate as carried out by GOT also decreased. Reduction of GOT activity suggests that less aspartate was formed, and as a consequence, amide export to aboveground parts also decreased. The absence of differences in PEPC and GOT-specific activities (data not shown) suggests that lower enzyme activities associated with elevated temperature and drought were a consequence of depleted protein content. These findings were further confirmed by TSP determinations. These limitations might explain the inhibitory effect of elevated temperature on nitrogen fixation. Factors that decrease photosynthate production in the host plant also decrease N2 . This is due to the fact that the rate of photosynthate supply to the nodule is one of the primary factors limiting N2 fixation (Vance and Heichel, 1991; Hunt and Layzell, 1993; De Luis et al., 1999). However, the absence of a temperature effect on TSS in nodule tissue in this experiment suggests that decreased N content was not caused by a reduction of carbohydrate supply. As Weisbach et al. (1996) showed, whole-nodule carbon metabolites are not a good indicator of nodule activity. The response of the nodule bacteroid fraction paralleled that of the plant fraction; elevated temperature negatively affected bacteroid enzyme activity regardless of water availability, although the inhibitory effect on the bacteroid fraction was even greater. The MDH activity decrease observed in plants exposed to elevated temperature suggests that malate
should also be reduced. Malate depletion implies that less carbon is redirected to mitochondria and that there is less energy available for N2 fixation. The observed enzyme activity decrease could be related to a reduction of the total soluble protein of the bacteroid. The large (70%) reduction in soluble protein could affect nitrogenase protein responsible for N2 fixation activity. These results show that the nodule bacteroid fraction was more sensitive to drought and elevated temperature than the nodule plant fraction. In summary, elevated temperature and low water availability constrained plant growth, particularly when plants were exposed to both stresses. Elevated temperature inhibited plant growth by diminishing net photosynthesis and nitrogen fixation. Exposure to 28 ◦ C reduced CO2 fixation rates due to its inhibitory effect on rubisco activity. Drought decreased plant growth through its inhibitory effect on leaf area formation in plants that otherwise had the same photosynthetic rates. Nitrogen fixation was also inhibited by elevated temperature, but in a different manner that was dependent on water status. The decrease recorded for well-watered plants was due to a reduction in nodule activity, whereas the decrease noted in plants grown under conditions of lower water availability was caused by a reduction of nodule activity and less nodule dry mass formation.
Acknowledgements This work was supported by the Spanish Science and Technology Ministry (BFI2000-0154; BFU2004-05096) and Fundaci´on Universitaria de Navarra. I. Aranjuelo was the recipient of a research grant from the Spanish Science and Technology Ministry (FP2000-52313). The technical cooperation of A. Urdiain and H. Richardson is acknowledged.
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Effect of elevated temperature and water availability on CO2 exchange and nitrogen fixation of nodulated alfalfa plants Iker Aranjuelo, Juan Jos´e Irigoyen, Manuel S´anchez-D´ıaz ∗ Departamento de Fisiolog´ıa Vegetal, Facultades de Ciencias y Farmacia, Universidad de Navarra, Irunlarrea s/n, 31008 Pamplona, Spain Received 21 December 2004; received in revised form 7 June 2005; accepted 31 October 2005
Abstract The aim of this study was to investigate the effects of predicted temperature increases and drought conditions of Mediterranean environments on N2 -fixing alfalfa plants. One-month-old plants inoculated with Sinorhizobium meliloti strain 102F78 were grown in growth chambers under different temperature (25/15 or 28/18 ◦ C, day/night) and water availability (control or drought) regimes. Elevated temperature and drought reduced plant dry mass and leaf area, especially when both stresses were combined. The inhibitory effect of elevated temperature on plant growth was a consequence of decreased CO2 and N2 fixation rates. A photosynthetic decrease resulted from the inhibition of rubisco activity, probably associated with a lower activation state. An absence of differences in photosynthesis in relation to water availability suggests that drought decreased plant growth due to its negative effect on leaf area. Rising temperature and drought affected the nitrogen content negatively, although effects differed. Elevated temperature inhibited nodule activity drastically, whereas the inhibitory effect resulting from drought centred on nodule dry mass (DM) production. Plants exposed to a combination of elevated temperature and drought were the most negatively affected. © 2005 Elsevier B.V. All rights reserved. Keywords: Alfalfa; Drought; Nitrogen; Nodule; Photosynthesis; Temperature
1. Introduction General circulation models (GCM) predict that ambient temperature in the Mediterranean basin is going to increase 1–3 ◦ C by the middle of this century. Rising temperature will increase evapotranspiration rates and exacerbate low water availability problems commonly observed in Mediterranean environments, in which current annual potential evapotranspiration is often nearly twice the amount of rainfall (S´abate et al., 2002). It has long been known that drought during the warmest period of the year is the major stress factor limiting plant species distribution and growth in Mediterranean regions of the world (Mooney, 1983). Most studies analysing the effect of Mediterranean environments on plant growth have considered temperature and water availability separately. However, it is well known that the effect of combined stresses on plant growth causes alterations that cannot ∗
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be predicted if they are analysed alone, such as those resulting from synergistic and antagonistic phenomena (Valladares and Pearcy, 1997). For example, Chaves and Pereira (2004) observed that although photochemical processes are very resistant to low water availability, a down regulation of the photosynthetic apparatus occurs when plants are exposed simultaneously to drought and elevated temperature conditions. In addition, we would like to highlight the observation of Kaiser (1987) suggesting that the particular way in which water stress is imposed might be of special importance not only in understanding the field response to drought, but also in evaluating the plant’s capacity to acclimate to stress. Withholding water is the most common method for short-term experiments, but sustained or cyclic water stress is needed to simulate more realistic responses to drought (Pennypacker et al., 1990). The inhibitory effects of elevated temperatures target photosynthesis, water status, and N2 fixation in the case of N2 -fixing plants (Bushby, 1982). Many publications show
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that the photosynthetic rates of evergreen species in Mediterranean environments during summer drought decrease due to stomatal closure (Pe˜nuelas et al., 1998; Llusi`a and Pe˜nuelas, 2000) and metabolic limitation (rubisco activity, RuBP regeneration, etc.) (Tretiach et al., 1997; Larcher, 2000). CO2 fixation may be affected by an altered excitation energy distribution associated with changes of thylakoid membrane fluidity and by damaged Calvin cycle enzyme activities. Enhanced CO2 /O2 solubility coupled with diminished specificity of rubisco for CO2 associated with plants grown under high temperature is reflected in enhanced photorespiration ¨ and decreased photosynthesis rates (Jordan and Ogren, 1984; Brooks and Farquhar, 1985). Besides, Stitt et al. (1991) explained reduced CO2 fixation rates as a consequence of lower photoassimilate requirements, which lead to carbohydrate accumulation and the inhibition of Calvin cycle enzyme activities. The sensitivity of legume-Sinorhizobium symbiosis to ambient stresses like temperature and low water availability is also well known (Antol´ın and S´anchez-D´ıaz, 1992; Serraj et al., 1999; Hungria and Vargas, 2000; Streeter, 2003; Curtis et al., 2004; Thomas et al., 2004). Furthermore, some authors (Castellanos et al., 1996; Thomas et al., 2004) suggest that the effect of water deficit on N accumulation and N2 fixation is larger than that on biomass accumulation. Alfalfa is a temperate forage frequently exposed to low water availability, N-deficient soils and high temperature conditions. Photosynthesis supplies organic carbon to nodules, where it is used by the nitrogenase enzyme in the bacteroid inside nodules as a source of energy and reducing power to fix N2 (Azc´on-Bieto et al., 2000). The products of N2 fixation, either amides or ureids, are exported to the plant via the xylem (Schubert et al., 1995; Walsh, 1995). Factors that increase photosynthesis increase N2 fixation, while factors decreasing photosynthesis tend to decrease N2 fixation. This coupling results in the regulation of nitrogenase activity in plants by photosynthesis (carbon supply), nitrogen availability (N source strength) and N demand (N sink strength). In order to investigate the deleterious effect of elevated temperature and drought on CO2 fixation and its relation with N2 assimilation, we analysed rubisco activity, carbohydrate concentration, and several nodule enzymes involved in C and N metabolism. Hexoses provided by the plant are hydrolysed in the nodule to obtain phosphoenolpyruvate through the glycolytic pathway. Phosphoenolpyruvate carboxylase (PEPC) then catalyses the combination of respiratory CO2 (as HCO3 − ) with phosphoenolpyruvate to produce oxaloacetate, which is converted to malate by malate dehydrogenase (MDH). This malate can either be used as a source of carbon and energy for bacteroid consumption, enter mitochondria to be oxidised in the tricarboxylic acid cycle, or contribute to ammonia assimilation in the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle (Cabrerizo et al., 2001). MDH also forms a complex with the glutamate oxaloacetate transaminase (GOT) enzyme. This complex is responsible for transamination between glutamate and
oxaloacetate to yield 2-oxoglutarate and aspartate, which is converted into asparagine by asparagine synthetase. A dicarboxylic acid like malate, whilst excluding other putative substrates like ␣-ketoglutarate, glutamate, pyruvate and arabinose, is a likely candidate to support N2 fixation in nodules (Udvardi and Day, 1997). Serraj et al. (1999) suggest that N2 fixation is also regulated by nodule permeability to O2 and feedback regulation by N compounds under conditions of low water availability. A lower rate of water movement out of the nodule during drought stress may restrict export of N2 fixation products, thus inhibiting nitrogenase activity via a feedback mechanism (Pate et al., 1969; Serraj et al., 1999). The optimum temperature range for root-nodule symbiosis is between 25 and 33 ◦ C (Pankhurst and Sprent, 1976), although reductions in N2 fixation activity have been observed above 28 ◦ C (Hungria and Franco, 1993). Elevated temperature might affect N2 fixation directly or indirectly. Direct inhibition by temperature is a consequence of decreased nodule development (Dart and Mercer, 1965; Piha and Munns, 1987), functionality (Meyer and Anderson, 1959; Piha and Munns, 1987; Hern´andez-Armenta et al., 1989) and accelerated nodule senescence. Indirect inhibition is related to temperature effects on root hair formation depression, reduction of nodulation sites (Frings, 1976; Jones and Tisdale, 1921), and modified adherence of bacteria to root hairs (Frings, 1976). The main objective of the present study is to investigate the effect of predicted temperature and low water availability conditions on N2 -fixing alfalfa plant growth. In order to analyse the effect of elevated temperature and drought on nodulated alfalfa plants, experiments were performed in growth chambers in which plants were exposed to conditions differing in temperature (25 ◦ C versus 28 ◦ C) and water availability (control versus drought). It was predicted that temperature and drought would reduce plant growth as a consequence of their negative effects on water status, gas exchange, and N2 fixation. In order to test such a hypothesis, the effects of elevated temperature and water availability on water status, gas exchange and nodule metabolism were analysed. Plant water status was evaluated by the measurement of leaf relative water content (RWC). Stomatal (leaf conductance and intercellular CO2 concentration) and non-stomatal (light capture and rubisco carboxylation activity) measurements were carried out to determine the effects of elevated temperature and drought on CO2 fixation rates. Nodule N2 fixation was analysed through the analysis of several nodule plant and bacteroid key enzymes involved in nodule C and N metabolism.
2. Material and methods 2.1. Plant material and experimental design Seedlings of alfalfa (Medicago sativa L. cv. Arag´on) were transferred into 2 L pots (20 plants per pot) containing
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a mixture of inert perlite and vermiculite (2/1, v/v). During the first month, plants were inoculated three times with Sinorhizobium meliloti strain 102F78 (The Nitragin Co., Milwaukee, WI, USA) and grown in a greenhouse at 25/15 ◦ C (day/night) with a photoperiod of 14 h under natural daylight, supplemented with fluorescent lamps (Sylvania DECOR 183, Professional-58W, Germany) that provided a photosynthetic photon flux density (PPDF) of about 300–400 mol m−2 s−1 . Plants were watered twice a week with Evans N-free nutrient solution (Evans, 1974) and once a week with tap water to avoid salt accumulation in pots. When 30 days old, plants were transferred to controlled environment chambers (Conviron PGV 36, Winnipeg, Canada) and randomly assigned to four treatments (eight pots per treatment): well-watered and water-stressed plants grown at a temperature of 28 ◦ C (elevated) or 25 ◦ C (control). Plants were maintained in growth chambers under the specified growth conditions for a period of 30 days, by the end of which plants were 60 days old. Pots were rotated every week from one chamber to another to avoid chamber effects. Well-watered plants were irrigated until a maximum soil volumetric water content (θ v ) of around 400 mm3 mm−3 was reached. The drought level used in treatments corresponded to 70% θ v of well-watered plants (around 280 mm3 mm−3 ). Such θ v levels were maintained throughout the experiment by measuring the transpired water daily (calculated by weighing the pots) and replenishing the lost water. To reduce soil evaporation, pots were covered with plastic sheets perforated with very small holes to allow stems to pass through. The desired drought level was reached around 12 days after the beginning of the treatment, when plants were 42 days old. Fully irrigated plants were alternatively watered with Evans N-free nutrient solution (Evans, 1974) and distilled water, whereas plants subjected to drought were always watered with Evans solution in order to supply all treatments with the same amount of nutrients. Conditions in the growth chamber involved temperatures of 25/15 ◦ C (day/night, low temperature) and 45% RH (1.7 kPa vapor pressure deficit, VPD) or 28/18 ◦ C (day/night, elevated temperature) and 54% RH (1.7 kPa VPD), ambient CO2 of around 400 mol mol−1 , a 14 h photoperiod and 600 mol m−2 s−1 PPFD. The experiment was repeated in two consecutive years. Experiments in the second year were performed to confirm results obtained in the first year. Values presented in this paper correspond to mean values of data collected for both years. Temperature was determined at leaf and root levels. Measurements corresponding to root levels were obtained from the soil surface and at a depth of 5 cm (where most of the nodules develop). Soil temperature was measured with a thermocouple thermometer. Leaf temperature was measured daily on adaxial and abaxial surfaces of the youngest fully expanded leaves, where gas exchange and chlorophyll fluorescence were measured. Leaf temperature was determined using a non-contact infrared thermometer LP-H (Raytec, Rynger PK, Berlin, Germany). Measurements were performed under growth conditions inside the growth chamber. Apical, fully
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expanded leaves without symptoms of injury were selected for each determination. 2.2. Growth parameters and water relations To record plant growth parameters, the first harvest was performed when the plants reached the desired θ v level (42day-old plants), and the second harvest was performed on the last day of each treatment (60-day-old plants). Plant organs were weighed separately for both harvests after being separated into leaves, stems, roots and nodules. The dry mass (DM) of each organ was obtained after drying in an oven at 80 ◦ C for 48 h. A root:shoot ratio was calculated as the proportion of root and nodule DM versus leaf and stem DM. Leaf area was measured directly using an automatic leaf area meter (Li-3000, LiCor, NE, USA). Plant water status was evaluated by measuring the relative water content of a leaf (Weatherley, 1950). Soil volumetric water content was calculated by weighing pots every day at the beginning of the photoperiod. Water use efficiency of productivity (WUEp ) is calculated as the ratio of produced dry mass to the water consumption in a period of time (Larcher, 1995; De Luis et al., 1999). WUEp was calculated for the period between the second harvest (42-day-old plants) and the final harvest (60-day-old plants). 2.3. Leaf determinations Fully expanded apical leaves from 50-day-old plants were individually enclosed in a leaf chamber (1010-M, Waltz, Effeltrich, Germany), and the gas exchange rate was measured with a portable photosynthesis system (HCM-1000, Waltz) under growth conditions. Net photosynthesis (A) and leaf conductance (g) were calculated as described by von Caemmerer and Farquhar (1981). Leaf internal CO2 concentration (Ci ) was estimated from net photosynthesis and conductance measurements according to Farquhar and Sharkey (1982). The optimal quantum yield of photosystem II was measured using a photosynthesis yield analyser (MINI-PAM, Waltz). Measurements were taken simultaneously with gas exchange determinations. Measuring conditions were the same as those of growth conditions, but the leaves were first dark-adapted for 20 min before carrying out the measurements. Leaf total soluble proteins (TSP), total soluble sugars (TSS) and starch concentration were quantified by grinding and filtering 100 mg of fresh weight frozen leaf tissue in a cold mortar using an extraction buffer containing 50 mM Kphosphate (pH 7.5). Extract was filtered and centrifuged at 28,710 × g for 15 min at 4 ◦ C. The supernatant was used for TSP and TSS quantification, whereas starch was measured in the pellet as described by Jarvis and Walker (1993). TSP levels were measured using the protein dye-binding method of Bradford (1976), while TSS determinations followed the procedure of Yemm and Willis (1954).
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Pigment concentration was determined in each leaf used for gas exchange and chlorophyll fluorescence analyses. Extracts for pigment analysis were prepared by grinding and filtering 100 mg fresh weight of the last fully developed leaves in a cold mortar with 10 ml of ethanol (95%, v/v). The homogenate was centrifuged at 3165 × g for 10 min at 4 ◦ C. The supernatant was used for pigment analyses. An aliquot of 1 ml from this extract was taken, 4 ml of ethanol 95% were added, and the absorbance measured at 750, 665, 649 and 470 nm. Absorbance determinations were carried out with a Spectronic 2000 (Bausch and Lomb, Rochester, USA) spectrophotometer. Extinction coefficients and equations used to calculate pigment contents were those described by Liechenthaler (1987). Rubisco (E.C. 4.1.1.39) extractable activity was determined in the same leaves used for gas exchange and chlorophyll fluorescence measurements. Enzyme activity was monitored by grinding and filtering 250 mg fresh weight of frozen leaf material, representing the last fully developed leaves, in a cold mortar using an extraction buffer containing 50 mM K-phosphate buffer (pH 7.5), 1 mM ethylenediaminetetraacetic acid iron(III) sodium salt (EDTA, Sigma–Aldrich), 8 mM MgCl2 ·6H2 O, 5 mM dl-dithiothreitol (freshly prepared, Sigma–Aldrich) and 1% (p/v) polyvinylpolypyrrolidone (PVPP, Sigma–Aldrich). The extract was then clarified by centrifugation at 26,890 × g for 10 min at 4 ◦ C. Enzyme activity in the extract was determined by measuring the absorbance at 340 nm, as described by Lilley and Walker (1974). 2.4. Nitrogen content and specific nodule activity Leaf, stem and root samples, previously dried at 60 ◦ C for 48 h, were ground in a mill with titanium blades and stored in vials placed in desiccators over silica gel. The nitrogen concentration was determined by means of sulphuric acid digestion in a B¨uchi K-424 (B¨uchi, Switzerland). Samples of 100 mg were digested by adding 20 ml H2 SO4 and a Kjeldhal Cu-Se catalytic pill. The digestion process ran for 1 h at 250 ◦ C until the sample was clarified. The samples were then diluted to 50 ml with distilled water. For the determination of nitrogen content, 5 ml of ionic strength adjuster (ISA, Ref. 951211, Orion, NY, USA) were added to 5 ml of measuring solution. Measurements were performed with an ammonia selective electrode (Orion 95-12BN, NY, USA) using 0.1 mM ammonium chloride as a standard. Specific nodule activity (SNA) was calculated as described by Brioua and Wheeler (1994), being the ratio between plant total nitrogen content and nodule DM. 2.5. Nodule determinations Five hundred milligrams of freshly harvested nodules were crushed in 10 ml of 50 mM K-phosphate buffer (pH 7.8), with 0.2% (v/v) 2-mercaptoethanol, 0.1 mM Na2 -EDTA and 10% (w/w) PVPP in a cold mortar. Isolation of plant and bac-
teroid fractions were performed as described by Irigoyen et al. (1992). To ensure no contamination between plant and bacterial fractions, phosphoenolpyruvate carboxylase was used as a plant fraction enzyme marker. No PEPC activity was detected for the bacteroid fraction. Phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) activity was assayed in nodule plant fractions by monitoring NADH oxidation spectrophotometrically. The reaction was performed as described by Deroche et al. (1983). Malate dehydrogenase (E.C. 1.1.1.37) and isocitrate dehydrogenase (ICDH, E.C. 1.1.1.42) enzymes were analysed in both plant and bacteroid fractions. The reaction medium and assay conditions used are detailed in Vance and Stade (1984). Malate dehydrogenase was measured by monitoring NADH oxidation at 340 nm, whereas ICDH was determined by recording the NADPH change at 340 nm. Glutamate oxaloacetate transaminase (E.C.2.6.1.1) was assayed only in the plant fraction, according to the method detailed in Bergmeyer (1974). Total soluble protein was determined in both plant and bacteroid fractions, whereas TSS was only determined in plant fractions. Total soluble protein and TSS determination on aliquots of nodule extraction were performed as described in the leaf determination section. 2.6. Statistical analysis Two factor analyses of variance (ANOVA, Sokal and Rohlf, 1986) at 0.5 and 0.1% levels were performed to partition the variance into main effects and an interaction between the two factors (temperature and drought). When the F-ratio was significant, least significant differences were evaluated using the Tukey b-test (P < 0.05).
3. Results The leaf temperature of plants grown at 28 ◦ C was higher than that of plants grown at 25 ◦ C for both irrigation treatments (Fig. 1). Drought did not alter leaf temperature (Fig. 1). The same response was observed at the substrate surface level and at a depth of 5 cm (Fig. 1). Total dry mass was negatively affected by elevated temperature (28 ◦ C) in both fully and partially watered treatments (Table 1). Drought also reduced plant production. Plants exposed to both elevated temperature and drought incurred the greatest decrease in plant production. Individual DM of leaves, shoots and roots was negatively affected by the combination of both stress factors. Leaf area was negatively affected by low water availability, whereas only temperature decreased leaf area in partially watered treatments. Elevated temperature did not change the root:shoot ratio, which increased in response to low water availability. Leaf relative water content was not affected by temperature, whereas drought conditions had a negative effect on leaf RWC (Table 1). Analysis of soil volumetric water content (Table 1) revealed that fully watered plants had a higher soil
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Table 2 The effect of elevated temperature and drought on net photosynthesis (A), rubisco extractable activity, leaf total soluble sugars (TSS) and starch concentration of nodulated alfalfa plants (mol m−2 s−1 )
A Rubisco (mol g−1 DM min−1 ) TSS (mg g−1 DM) Starch (mg g−1 DM)
C25
C28
D25
D28
13.2a 20.0a 80.0ab 94.7b
8.4b 10.9b 53.1c 131.4a
14.1a 18.8a 86.0a 80.7b
10.5b 12.2b 64.6bc 99.9b
Each row represents the mean of 12 plants. Other details as in Table 1.
Fig. 1. The effect of elevated temperature and drought on leaf (adaxial) and soil (surface and 5 cm depth) temperature of nodulated alfalfa plants. Unshaded bars correspond to fully watered treatments; shaded bars correspond to partially watered plants. Each value represents the mean of 16 plants. Means with identical letters are not significantly different (P > 0.05).
water availability (≈30% more) when compared with partially watered plants, irrespective of temperature treatment. Although water use efficiency of productivity values were not changed significantly by temperature or water availability (Table 1), further analyses revealed that plants grown at 28 ◦ C produced less DM with lower water requirements. Plants grown at 28 ◦ C under fully and partially watered conditions suffered a reduction in net photosynthetic rate
(Table 2). CO2 fixation was not affected by water availability. There was no significant effect of temperature and drought on leaf conductance or intercellular CO2 concentration, and grand mean values were 108.2 mmol m−2 s−1 and 207.2 mol mol−1 , respectively. Rubisco extractable activity results were similar (Table 2) to observed photosynthesis measurements. Enzyme total activity was inhibited by elevated temperature, whereas no effect due to water availability was detected. It should be highlighted that there was no significant effect by temperature or water availability on total leaf soluble protein concentration (grand mean value of 70.3 mg g−1 DM). Elevated temperature decreased TSS (Table 2). No significant differences in starch concentration were detected between treatments, except for increased values recorded in fully watered plants grown under elevated temperature (Table 2). Leaf photosynthetic pigment content analyses showed that there was no appreciable change induced by temperature and/or water availability. Grand mean values for ChlA , ChlB , ChlA+B and Cx+c were 9.2, 2.9, 12.1 and 2.3 mg g−1 DM, respectively. There were no significant differences of Fv /Fm between treatments, which displayed a grand mean value of 0.8. Plant nitrogen content was inhibited by both temperature and water availability (Fig. 2). The interaction between elevated temperature and drought resulted in a 75% reduction of the N content. Specific nodule activity was significantly
Table 1 The effect of elevated temperature and drought on total dry mass (DM), leaf DM, stem DM, root DM, nodule DM, leaf area and root:shoot ratio, leaf relative water content (RWC), volumetric soil water content (θ v ), water use efficiency of productivity (WUEp ) and its components, DM production and water consumption of nodulated alfalfa plants Total DM (g plant−1 ) Leaf DM (g plant−1 ) Stem DM (g plant−1 ) Root DM (g plant−1 ) Nodule DM (mg plant−1 ) Leaf area (cm2 ) Root:shoot (g g−1 ) RWC (%) θ v (mm3 mm−3 ) WUEp (mg DM g−1 H2 O) DM production (g DM plant−1 ) Water consumption (g H2 O plant−1 )
C25
C28
D25
D28
2.2a 0.5a 0.7a 0.9a 39a 91.2a 0.8b 87.2a 480a 2.8a 2.0a 710.9a
1.3b 0.3b 0.4b 0.5b 43a 66.3ab 0.7b 84.7a 470a 2.6a 1.2b 451.0b
1.3b 0.3b 0.3b 0.7a 23b 43.9b 1.4a 76.0b 340b 3.1a 1.1b 357.3c
0.5c 0.1c 0.1c 0.2c 16b 26.9c 1.2a 76.2b 310b 2.1a 0.3c 165.9d
C25 and C28: fully watered plants grown under control (25 ◦ C) and elevated (28 ◦ C) temperature; D25 and D28: partially watered plants grown under control (25 ◦ C) and elevated (28 ◦ C) temperature. Each row represents the mean of 40 plants, except for RWC, which represents the mean of 12 plants. Means with identical letters (a–d) are not significantly different (P > 0.05).
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Fig. 2. The effect of elevated temperature and drought on plant total nitrogen content and specific nodule activity (SNA) of nodulated alfalfa plants. Each column represents the mean of 12 samples. Other details as in Fig. 1.
lower in elevated temperature treatments (28 ◦ C), whereas no significant effect was observed under conditions of low water availability (Fig. 2). Nodule plant fraction enzyme analyses showed that there were no differences on MDH activity between treatments, except for enhanced activity in fully watered plants grown under elevated temperature. An analysis of the separate effect of either temperature or water availability on GOT activity revealed that under optimal water availability conditions elevated temperature enhanced GOT activity whereas under low water availability conditions elevated temperature had inhibitory effect. The interaction of both stress factors reduced GOT activity significantly. Drought and elevated temperature alone did not affect PEPC activity, but PEPC enzyme activity decreased in plants exposed to both stress factors. An analysis of bacteroid fraction enzyme activity revealed that elevated temperature and drought inhibited MDH activity, especially when both parameters were combined (Table 3). There were no differences in ICDH activity between treatments. Nodule plant and bacteroid protein (Fig. 3) determinations showed that TSP content was neg-
Fig. 3. The effect of elevated temperature and drought on nodule bacteroid and plant total soluble protein (TSP) concentration of nodulated alfalfa plants. Each column represents the mean of 12 samples. Other details as in Fig. 1.
atively affected by elevated temperature in both nodule fractions. Low water availability decreased only the TSP in plant fractions. Total soluble sugars determinations did not reveal differences between treatments, and the grand mean value was 50.9 mg g−1 DM.
4. Discussion 4.1. Growth parameters and water relations Both elevated temperature and drought may limit plant growth (Aguirreolea and S´anchez-D´ıaz, 1989; Antol´ın and S´anchez-D´ıaz, 1992). Our study demonstrated that elevated temperature had a significant negative impact on biomass production, particularly when combined with drought. Increased temperature and drought reduced plant growth in a similar way (53 and 52%, respectively). Plants exposed to 28 ◦ C and drought were the most negatively affected (64% DM reduction). When analysing the effect of both stress factors on the different plant organs separately, a result similar
Table 3 The effect of elevated temperature and drought on nodule plant fraction malate dehydrogenase (MDH), glutamate oxaloacetate transaminase (GOT), phosphoenolpyruvate carboxylase (PEPC), isocitrate dehydrogenase (ICDH) and bacteroid fraction MDH and ICDH activities of nodulated alfalfa MDH (mmol g−1 DM min−1 ) GOT (mol g−1 DM min−1 ) PEPC (mol g−1 DM min−1 ) ICDH (mol g−1 DM min−1 ) MDH (mol g−1 DM min−1 ) ICDH (mol g−1 DM min−1 )
C25
C28
D25
D28
0.6b 124b 34a 87a 41a 1.5a
1.6a 150a 33a 19b 22b 1.0a
0.9b 129b 32a 17b 30b 1.7a
0.8b 96c 20b 13b 18c 1.0a
Each row represents the mean of 12 plants. Other details as in Table 1.
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to that of total DM is observed: elevated temperature and drought reduced leaf, shoot and root formation. Nodule dry mass production was not affected by increased temperature; however, low water availability had an inhibitory effect. Analyses of the root:shoot ratio revealed that the temperature increase affected above and belowground systems in a similar way. Plants subjected to drought had a larger root:shoot ratio, resulting mainly from a larger root biomass. Several authors (Hilbert and Canadell, 1995; Lloret et al., 1999) have hypothesized that this response may be an adaptation to the Mediterranean dry summer season. Variations in dry mass production observed between plant organs at elevated temperature could be explained by differences in sensitivity to high temperature (Sutcliffe, 1980). As shown with DM, leaf area was sensitive to increased temperature and drought. The absence of differences in CO2 fixation rates between fully and partially watered plants suggests that the inhibitory effect of low water availability on plant growth was centred on the leaf surface. Drought conditions and reduced leaf surface area resulted in decreased photosynthetic rates at the whole plant level. Diminished leaf area in plants subjected to drought could be the result of reduced cell division and elongation growth (Legg et al., 1975; Boyer et al., 1985), two processes extremely dependent on water availability. Leaf relative water content measurements revealed that differences observed in plant growth were due to temperature alone and not to an unequal water status, as the soil and leaf water contents were similar. According to Craufurd et al. (1999), heat stress might reduce WUE through the deleterious effect of temperature on carboxylation and respiration processes, and through alterations on water consumption. Although WUEp was not modified by temperature and water availability, analyses of WUEp components showed that they were affected negatively by both stress factors. Decreased water consumption of plants with the same leaf conductance is explained by their lower leaf surface, particularly in partially watered plants grown at 28 ◦ C. The absence of differences in WUEp could be explained by the fact that temperature and drought decreased DM production and water consumption simultaneously. 4.2. Leaf determinations As mentioned previously, the negative effects of elevated temperature and drought on alfalfa growth can be linked to changes in photosynthetic rates. Net photosynthesis can be diminished by stomatal limitations (associated with stomatal opening and closure) or non-stomatal limitations, generally corresponding to inhibitions of the photosynthetic apparatus (Escalona et al., 1999). Leaf conductance and intercellular CO2 concentration were unaffected by increased temperature or drought, which means that photosynthetic inhibition in plants grown at 28 ◦ C did not result from stomatal limitations. Although some authors report that stomata close at higher temperature, the closure probably results from a stomatal response to VPD that normally occurs when the
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leaf temperature is increased. As Berry and Bj¨orkman (1980) observed, when the same VPD is maintained, as was the case in this experiment, the stomata of plants exposed to elevated temperature remain open. Berry and Bj¨orkman (1980) also noted that stomata of plants grown under elevated temperature remained open at temperatures that cause damage to the photosynthetic apparatus. Decreased carboxylation efficiency might be a consequence of damage to photochemical reactions (Fellers et al., 1998). There may be a higher probability for photo-damage under limiting growth conditions (Baroli and Melis, 1998). The results of this study show that the optimum quantum yield of photosystem II, chlorophyll and carotenoid concentrations were within normal ranges, and that inhibition of photosynthesis was not caused by photochemical damage. These results are consistent with those of other studies (Oliveira and Pe˜nuelas, 2001; Ogaya and Pe˜nuelas, 2003) conducted in the Mediterranean basin, which showed that photoinhibition of PSII under drought conditions was only detected during the cold winter, and that no effect was detected in summer. Leegood and Edwards (1996) suggest that the mechanisms for reduced photosynthetic metabolism in C3 plants in response to increased temperature involve the enhancement of photorespiration and the inactivation of rubisco. The absence of differences in leaf respiration (data not shown) suggests that the decreased photosynthetic efficiency was a consequence of altered rubisco activity, as confirmed by rubisco extractable activity measurements. This reduction could be caused by the inactivation of the enzyme or by a reduction of enzyme concentration. The absence of differences in leaf total soluble protein suggests that decreased rubisco activity could be due to enzyme inactivation. According to several authors (Weis, 1981; Fellers et al., 1998), rubisco activity is regulated by rubisco activase. Some authors (Fellers et al., 1998; Escalona et al., 1999) have found that rubisco activase activity is sensitive to elevated temperature. Increased temperature may have inhibited rubisco activase, which would have reduced rubisco activity. It is also noteworthy that photosynthesis was not affected by water availability. In accordance with results obtained by other authors (De Luis, 2000; Ogaya and Pe˜nuelas, 2003), drought treatments tended to present net photosynthetic rates per soil water content that were higher than those of control treatments. This result indicates a quick and incipient acclimation to continuous low soil water availability, and suggests that plant growth is more sensitive than photosynthesis to low water availability regimes (Kramer and Boyer, 1995). 4.3. Nitrogen content and specific nodule activity As mentioned in the introduction, N2 -fixing plants such as legumes might also experience decreased plant growth due to the inhibitory effects of elevated temperature and drought on N2 fixation. The observed reduction in total nitrogen content in plants exposed to elevated temperature and low water availability can be explained by a decreased nodule dry mass
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formation and/or by diminished nodule activity. Specific nodule activity was analysed in an effort to determine which of these factors limited nitrogen fixation. The absence of an effect by drought on SNA indicates that low water availability inhibited N2 fixation through its deleterious effect on nodule DM production. These results accord with those obtained from Serraj and Sinclair (1998) through experiments performed in greenhouse and field conditions. Furthermore, the analysis of SNA also revealed that the decreased N content observed in plants grown at 28 ◦ C was caused by the negative effect of elevated temperature on nodule activity. 4.4. Nodule determinations To confirm previous observations, analyses were performed in plant and bacteroid fractions of several key nodule enzymes involved in nodule C and N metabolism, together with TSP, TSS and starch content. A general overview of nodule plant fraction metabolism results shows that drought did not modify enzyme activity. Furthermore, the plant fraction was more sensitive to elevated temperature, particularly under conditions of low water availability. Plants exposed to elevated temperature and drought had lower PEPC and GOT activities. PEPC provides up to 25% of the carbon required for nitrogenase activity and N assimilation (King et al., 1986). Reduction of PEPC suggests that the nodules of plants grown under conditions of elevated temperature and low water availability had an inadequate C supply. Furthermore, reduced PEPC activity also implies that less oxaloacetate was formed, and therefore the transamination between glutamate and oxaloacetate as carried out by GOT also decreased. Reduction of GOT activity suggests that less aspartate was formed, and as a consequence, amide export to aboveground parts also decreased. The absence of differences in PEPC and GOT-specific activities (data not shown) suggests that lower enzyme activities associated with elevated temperature and drought were a consequence of depleted protein content. These findings were further confirmed by TSP determinations. These limitations might explain the inhibitory effect of elevated temperature on nitrogen fixation. Factors that decrease photosynthate production in the host plant also decrease N2 . This is due to the fact that the rate of photosynthate supply to the nodule is one of the primary factors limiting N2 fixation (Vance and Heichel, 1991; Hunt and Layzell, 1993; De Luis et al., 1999). However, the absence of a temperature effect on TSS in nodule tissue in this experiment suggests that decreased N content was not caused by a reduction of carbohydrate supply. As Weisbach et al. (1996) showed, whole-nodule carbon metabolites are not a good indicator of nodule activity. The response of the nodule bacteroid fraction paralleled that of the plant fraction; elevated temperature negatively affected bacteroid enzyme activity regardless of water availability, although the inhibitory effect on the bacteroid fraction was even greater. The MDH activity decrease observed in plants exposed to elevated temperature suggests that malate
should also be reduced. Malate depletion implies that less carbon is redirected to mitochondria and that there is less energy available for N2 fixation. The observed enzyme activity decrease could be related to a reduction of the total soluble protein of the bacteroid. The large (70%) reduction in soluble protein could affect nitrogenase protein responsible for N2 fixation activity. These results show that the nodule bacteroid fraction was more sensitive to drought and elevated temperature than the nodule plant fraction. In summary, elevated temperature and low water availability constrained plant growth, particularly when plants were exposed to both stresses. Elevated temperature inhibited plant growth by diminishing net photosynthesis and nitrogen fixation. Exposure to 28 ◦ C reduced CO2 fixation rates due to its inhibitory effect on rubisco activity. Drought decreased plant growth through its inhibitory effect on leaf area formation in plants that otherwise had the same photosynthetic rates. Nitrogen fixation was also inhibited by elevated temperature, but in a different manner that was dependent on water status. The decrease recorded for well-watered plants was due to a reduction in nodule activity, whereas the decrease noted in plants grown under conditions of lower water availability was caused by a reduction of nodule activity and less nodule dry mass formation.
Acknowledgements This work was supported by the Spanish Science and Technology Ministry (BFI2000-0154; BFU2004-05096) and Fundaci´on Universitaria de Navarra. I. Aranjuelo was the recipient of a research grant from the Spanish Science and Technology Ministry (FP2000-52313). The technical cooperation of A. Urdiain and H. Richardson is acknowledged.
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