Blackwell Science, LtdOxford, UK PCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2001 2410October 2001 744 Growth condition effects on thermotolerance-enhancing compounds D. T. Hanson & T. D. Sharkey Original ArticleBEES SGML
Plant, Cell and Environment (2001) 24, 929–936
Effect of growth conditions on isoprene emission and other thermotolerance-enhancing compounds D. T. HANSON1 & T. D. SHARKEY2 1Australian
National University, Molecular Plant Physiology, Research School of Biological Sciences, PO Box 475, Canberra, Australian Capital Territory 2601, Australia and 2Department of Botany, University of Wisconsin-Madison, 430 Lincoln Drive, Madison, WI 53706-1381, USA
ABSTRACT Isoprene is emitted from the leaves of many plants in a light-dependent and temperature-sensitive manner. Plants lose a large fraction of photo-assimilated carbon as isoprene but may benefit from improved recovery of photosynthesis following high-temperature episodes. The capacity for isoprene emission of plants in natural conditions (assessed as the rate of isoprene emission under standard conditions) varies with weather. Temperaturecontrolled greenhouses were used to study the role of temperature and light in influencing the capacity of oak leaves for isoprene synthesis. A comparison was made between the capacity for isoprene emission and the accumulation of other compounds suggested to increase thermotolerance of photosynthesis under two growth temperatures and two growth light intensities. It was found that the capacity for isoprene emission was increased by high temperature or high light. Xanthophyll cycle intermediates increased in high light, but not in high temperature, and the chloroplast small heat-shock protein was not expressed in any of the growth conditions. Thus, of the three thermotoleranceenhancing compounds studied, isoprene was the only one induced by the temperature used in this study. Key-words: Quercus; heat shock; oak; xanthophyll.
INTRODUCTION The emission of isoprene from plant leaves is the largest known source of non-methane volatile organic hydrocarbons in the atmosphere (Guenther et al. 1995; Geron et al. 2000). Many plants produce isoprene and this appears to increase the recovery of photosynthesis following short, high-temperature episodes (Sharkey & Singsaas 1995; Singsaas et al. 1997; Sharkey, Chen & Yeh 2001). Thylakoid membranes and photosystem II (PSII) are particularly heat-sensitive components of photosynthesis and are easily disrupted by short periods of high temperature (Berry & Björkman 1980; Weis & Berry 1988; Havaux 1993; Bukhov et al. 1999). It is hypothesized that isoprene stabilizes thylakoid membranes during these short periods of high-
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temperature stress (Singsaas et al. 1997; Sharkey et al. 2001). The degree to which isoprene increases thermotolerance is dependent on the amount of isoprene present in the leaf (Singsaas et al. 1997). However, isoprene is not stored in the leaf (Delwiche & Sharkey 1993), so the rate of isoprene synthesis must increase with leaf temperature if it is to protect photosynthesis at high temperatures. Isoprene synthesis responds very rapidly to temperature increases as expected if it is important for short high-temperature episodes (Sanadze & Kursanov 1966; Rasmussen & Jones 1973; Tingey et al. 1979; Monson & Fall 1989; Loreto & Sharkey 1990; Sharkey & Loreto 1993; Singsaas & Sharkey 1998). Over longer time frames, the capacity for isoprene emission (determined by measuring the rate of emission under standard assay conditions of 30 ∞C and 1000 mmol photons m-2 s-1) is known to vary throughout the growing season and between sun- and shade-adapted leaves (Sharkey, Loreto & Delwiche 1991; Monson et al. 1992; Sharkey & Loreto 1993; Litvak et al. 1996; Sharkey et al. 1996; Sharkey et al. 1999). Attempts to correlate weather patterns with the isoprene emission capacity of oak trees in the field indicate that light and temperature are important factors in predicting capacity (Sharkey et al. 1999; Geron et al. 2000). Because leaf temperature is influenced heavily by light level (Roden & Pearcy 1993), it is not clear whether the effect of light is direct or indirect through its effect on leaf temperature. Xanthophyll cycle intermediates are also involved in thermotolerance. High-light conditions cause the deepoxidation of violaxanthin to antheraxanthin and zeaxanthin. The increase in zeaxanthin stabilizes thylakoid membranes by decreasing membrane fluidity (Havaux 1998). This stabilization increases the thermotolerance of thylakoid membranes that have been heat-treated for short periods (Havaux 1993; Havaux et al. 1996; Tardy & Havaux 1997). Antheraxanthin and zeaxanthin formed by rapid de-epoxidation of violaxanthin are better known for their protection against damage from excess light energy (reviewed in Demmig-Adams & Adams 1996; Gilmore 1997). Increased pool size and de-epoxidation status of xanthophyll cycle intermediates in leaves from high versus low light have been well documented (Thayer & Björkman 1990; Demmig-Adams & Adams 1992). However, changes in the pool size of xanthophyll intermediates in response to the temperature of growth conditions has mostly been 929
930 D. T. Hanson & T. D. Sharkey examined with respect to cold acclimation in high light, with a few studies also examining warm acclimation (Adams et al. 1995a; Adams, Hoehn & Demmig-Adams 1995b; Koroleva, Thiele & Krause 1995; Verhoeven, Adams & DemmigAdams 1996; Krause, Carouge & Garden 1999; Verhoeven, Adams & Demmig-Adams 1999). In addition to isoprene and xanthophylls, several heat-shock proteins accumulate in vegetative tissues in response to heat stress and some are thought to enhance thermotolerance (Vierling 1991; Waters, Lee & Vierling 1996). However, only the small chloroplast heat-shock protein has been demonstrated to protect PSII from high-temperature damage (Heckathorn et al. 1998). Light-induced expression has also been documented for some heat-shock proteins (Kropat et al. 1997), but not for the small chloroplast heat-shock protein. To test the effects of light and temperature on isoprene emission, xanthophyll intermediates and the chloroplast small heat-shock protein, oak seedlings were grown in four different conditions (high and low light at two temperatures). The conditions were intended to maximize the differences in isoprene emission without subjecting the plants to extreme levels of stress. We then assessed the capacity of the leaves to emit isoprene, measured xanthophyll cycle intermediates and looked for the induction of the chloroplast small heat-shock protein.
MATERIALS AND METHODS Plant material Two-year-old red oak (Quercus rubra L.) and white oak (Quercus alba L.) saplings (Musser Forests, Inc., Indiana, PA, USA) were grown in temperature-controlled greenhouses in the Biotron facility at the University of Wisconsin-Madison. There were no obvious differences between the red and white oak responses so the data for the two species were combined. Measurements were made on leaves that had completed expansion during the 7 weeks of acclimation to each growth condition. A single, mature leaf was measured on each sapling used in this study. The saplings were planted in 8 L pots using a commercial soilless peat mix (Metro-Mix 360; W.R. Grace and Co., Cambridge, MA, USA) and watered daily with one-quarter-strength Mir-Acid (Sterns Miracle Grow Products, Inc., Port Washington, NY, USA). Two high-ceiling greenhouse rooms were used. The cold room had day/night temperatures of 20/14 ∞C and the warm room had day/night temperatures of 32/24 ∞C. Room temperatures were allowed to drift between day and night temperatures for 1 h at the beginning and end of the photoperiod. This resulted in a gradual change in the room temperature rather than a sudden step-change. As the plants were grown during autumn and winter months, supplemental lighting was provided all day using high-pressure sodium bulbs. This provided approximately 300 mmol photons m-2 s-1 at leaf level in the unshaded conditions and maintained a 15 h day length. In half of each room, shade cloth was installed above the plants and below the lamps.
This reduced the light intensity at leaf level (averaged over the entire day) from 500 mmol photons m-2 s-1 in the sun conditions to 130 mmol photons m-2 s-1 in the shade conditions. Peak light intensities were around 1000 mmol photons m-2 s-1 in the sun and around 300 mmol photons m-2 s-1 in the shade. All oak trees grew actively under all four conditions, as evidenced by repeated production of new leaves and buds and by stem elongation.
Micrometeorology Light levels were monitored with a gallium arsenide photodiode (part number G1118; Hamamatsu Corp., Bridgewater, NJ, USA) above a single sapling in each growth condition. Air temperature was measured with a shaded thermocouple. The difference between leaf and air temperature was measured with a second thermocouple (Fig. 1) on a leaf near the air temperature thermocouple. One junction of the thermocouple was pressed against the lamina of a leaf by threading it through two nearby veins on the abaxial surface of the leaf. The other junction was suspended just below the leaf so that both junctions were in the same radiation environment. Leaf temperature was determined by adding the air temperature to the difference between leaf and air temperature. Thermocouples were chromel–constantan 3 mil (0·0762 mm) wire with spot-welded junctions. One leaf- and one air-temperature thermocouple was used per growth condition. Light and temperature measurements were collected once per minute with a CR-10 data logger and AM32 multiplexer from Campbell Scientific (Logan, UT, USA).
Figure 1. Thermocouple set-up for directly measuring the difference between leaf temperature and air temperature. One junction of the thermocouple was pressed against the lamina of a leaf by threading it through two nearby veins on the abaxial surface of the leaf. The other junction was suspended just below the leaf so that the leaf would shade the junction from direct solar radiation. The constantan wire between the two junctions was spot-welded to chromel wire to create the thermocouple junction. The chromel wires were connected to copper wire, which was attached to the data logger. The chromel–copper junctions were insulated to ensure that both junctions were at the same temperature. © 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 929–936
Growth condition effects on thermotolerance-enhancing compounds 931
Gas exchange and isoprene measurement Photosynthesis was measured using a portable gas exchange system with a red- and blue-light-emitting diode light source (Li-Cor 6400; Lincoln, NE, USA). Photosynthesis was measured simultaneously with the measurement of the capacity for isoprene emission under standard conditions of 30 ∞C and 1000 mmol photons m-2 s-1. Air entering the gas exchange system was drawn in from outside the greenhouses. Isoprene was detected with a fast isoprene sensor consisting of an ozone generator and a photomultiplier (Hills Scientific, Boulder, CO, USA) (Hills & Zimmerman 1990). The air exiting the gas exchange system was combined with a 0·8 standard litres per metre (SLPM) flow of 2% ozone produced by the ozone generator. Isoprene in the air stream reacts with ozone to produce an activated aldehyde. When the activated electron falls to the ground state, a photon is released and counted with the photomultiplier. Data were collected every second by a computer and 30 s averages were used for each measurement. Ambient levels of isoprene were measured before and after each leaf measurement and the average of the two ambient measurements was subtracted from the leaf measurement. The isoprene analyser was calibrated each day with a six-point standard diluted from a 6 ppmv isoprene standard in nitrogen (Scott-Martin Specialty Gases, Riverside, CA, USA).
Analysis of xanthophyll cycle intermediates and other pigments After measuring the capacity for isoprene in all four conditions, leaf punches (1·7 cm2) were taken from the same mature leaves used for isoprene measurements, placed in a foil pouch and immediately frozen in liquid nitrogen, then stored at -80 ∞C until analysis. Sample collection from all four conditions was completed within a period of about 1 h during sunny conditions in the afternoon. Photosynthetic pigments were extracted from the punches in acetone and quantified as described in Sharkey et al. (1996) and Thayer & Björkman (1990). After the pigments were extracted, the remaining pellet was frozen in liquid nitrogen and stored at -80 ∞C for later use in heat-shock protein detection.
Western blot analysis Protein pellets frozen after the acetone extraction of photosynthetic pigments were re-suspended by vortexing in lithium dodecyl sulfate (LDS) sample buffer (catalog number NP007, Novex, Inc., San Diego, CA, USA). These samples were boiled for 5 min before applying equal volumes to NuPAGE 4–12% Bis-Tris gel (NOVEX) with NuPAGE 2(N-morpholino) ethanesulfonic acid (MES) sodium dodecyl sulphate running buffer (Novex catalog number NPO323, NP002). Gel electrophoresis and Western transfer were conducted with the Novex XCell II Mini-Cell and blot module (catalog number EI9002 kit) according to the manufacturer’s directions. Proteins were transferred to a nitrocellulose membrane. © 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 929–936
The rabbit antibodies used were raised against a synthetic peptide covering the methionine-rich region of the small, chloroplast heat-shock protein (Downs et al. 1998; Heckathorn et al. 1998). This region is unique to the small, chloroplast heat-shock protein and is highly conserved among all land plants except bryophytes (Waters et al. 1996; Waters & Vierling 1999). The ECL western blotting system (catalog number RP2108, Amersham Pharmacia Biotech, Piscataway, NJ, USA) was used according to the manufacturer’s directions for antibody detection. Comparisons of acetone- and non-acetone-extracted, heat-shocked and non-heat-shocked white oak leaves showed no difference in the detection of the small, chloroplast heat-shock protein using this antibody (data not shown).
Statistical analyses SYSTAT 10·0 (SPSS, Chicago, IL, USA) was used to determine statistical significance of light and temperature effects via an analysis of variance with a Tukey HSD post hoc test for multiple comparisons. The data analysis add-in from Microsoft Excel 2000 (Microsoft Corp., Redmond, WA, USA) was used to calculate Student’s t-tests for determining the significance of differences between individual pairs of growth conditions.
RESULTS Leaf temperature and light intensity variation Leaf-level light intensities and leaf temperatures were affected by variation in cloud cover during the 7 week acclimation period (Fig. 2). A two-fold variation in light levels occurred between sunny and overcast days but shaded conditions were always approximately 25% of the light level in the sun conditions (Fig. 2a,b). Differences between light levels in cold and warm conditions (within sun and shade treatments) are due to the location and slight variation in orientation of photodiodes within each room and do not represent actual differences in growth conditions. Within a light condition (sun or shade), leaf temperatures generally differed by about 10 ∞C between warm and cold conditions (Fig. 2c,d). Leaf temperatures in the coldsun conditions were always around 4 ∞C higher than in the cold-shade conditions. On sunny days, leaf temperature was about 3 ∞C higher in warm-sun conditions than warm-shade conditions. Peak leaf temperatures frequently exceeded 40 ∞C in warm-sun conditions on sunny days. Peak leaf temperatures generally occurred prior to peak light intensity because of increases in cooling by the air handling system during mid-day. The maximum leaf temperature recorded during the 7 weeks was 43 ∞C in the warm-sun conditions and 39 ∞C in the warm-shade.
Effect of growth conditions on photosynthesis Photosynthetic rates (mmol CO2 m-2 s-1) were variable but there was no effect due to growth condition on photosyn-
Overcast day
Leaf temperature (°C)
PAR (mmol m–2 s–1)
Sunny day 1000
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(b)
800 600 400 200 0 40
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Capacity for isoprene emission (nmol m–2 s–1)
932 D. T. Hanson & T. D. Sharkey
20
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WSU WSH CSU CSH
20 10 0
1200 1300 1400
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Figure 4. Capacity for isoprene emission in all four growth conditions. Abbreviations and growth conditions are the same as in Fig. 2. Error bars represent standard error, n = 3 for warm-sun and n = 5 for the other three conditions.
Time of day (h)
Figure 2. Comparison of leaf-level light intensity and leaf temperatures between a sunny day and a completely overcast day in all four conditions. Measurements were recorded once per minute and the data shown is for the four peak hours of each day. WSU, warm and sun conditions (); WSH, warm and shaded conditions (); CSU, cold and sun conditions (); CSH, cold and shaded conditions (). Daytime greenhouse room temperatures were 32 ∞C in warm conditions and 20 ∞C in cold conditions.
Photosynthetic rate (mmol co2 m–2 s–1)
thesis (P = 0·905, Fig. 3) when measured at 30 ∞C and 1000 mmol photons m-2 s-1. No significant differences were found between sun and shade (P = 0·793) or warm and cold (P = 0·529). In addition, there were no significant differences between growth conditions when photosynthetic rates were expressed on a per chlorophyll a basis (data not shown).
3
Effect of growth conditions on the capacity for isoprene emission The capacity for isoprene emission in the warm-sun conditions was about two times the capacity in the warm-shade and the cold-sun, and over four times the capacity in the cold-shade (Fig. 4). Pooling the data by temperature or by light condition indicated that the capacity in warm conditions was twice the capacity in cold conditions (P = 0·008) and plants grown in the sun nearly three times the capacity of those grown in the shade (P < 0·001). The overall effect of growth condition on capacity was highly significant (P < 0·001). Warm-sun rates were significantly greater than all other conditions (P-values
