Planta

, Volume 220, Issue 3, pp 486–497

Enhanced thermotolerance of photosystem II in salt-adapted plants of the halophyte Artemisia anethifolia

Authors

  • Xiaogang Wen
    • Photosynthesis Research Centre, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of BotanyChinese Academy of Sciences
  • Nianwei Qiu
    • Photosynthesis Research Centre, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of BotanyChinese Academy of Sciences
  • Qingtao Lu
    • Photosynthesis Research Centre, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of BotanyChinese Academy of Sciences
    • Photosynthesis Research Centre, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of BotanyChinese Academy of Sciences
Original Article

DOI: 10.1007/s00425-004-1382-7

Cite this article as:
Wen, X., Qiu, N., Lu, Q. et al. Planta (2005) 220: 486. doi:10.1007/s00425-004-1382-7
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Abstract

Thermotolerance of photosystem II (PSII) in leaves of salt-adapted Artemisia anethifolia L. plants (100–400 mM NaCl) was evaluated after exposure to heat stress (30–45°C) for 30 min. After exposure to 30°C, salt adaptation had no effects on the maximal efficiency of PSII photochemistry (Fv/Fm), the efficiency of excitation capture by open PSII centers (Fv′/Fm′), or the actual PSII efficiency (ΦPSII). After pretreatment at 40°C, there was a striking difference in the responses of Fv/Fm, Fv′/Fm′ and ΦPSII to heat stress in non-salt-adapted and salt-adapted leaves. Leaves from salt-adapted plants maintained significantly higher values of Fv/Fm, Fv′/Fm′ and ΦPSII than those from non-salt-adapted leaves. The differences in Fv/Fm, Fv′/Fm′ and ΦPSII between non-salt-adapted and salt-adapted plants persisted for at least 12 h following heat stress. These results clearly show that thermotolerance of PSII was enhanced in salt-adapted plants. This enhanced thermotolerance was associated with an improvement in thermotolerance of the PSII reaction centers, the oxygen-evolving complexes and the light-harvesting complex. In addition, we observed that after exposure to 42.5°C for 30 min, non-salt-adapted plants showed a significant decrease in CO2 assimilation rate while in salt-adapted plants CO2 assimilation rate was either maintained or even increased to some extent. Given that photosynthesis is considered to be the physiological process most sensitive to high-temperature damage and that PSII appears to be the most heat-sensitive part of the photosynthetic apparatus, enhanced thermotolerance of PSII may be of significance for A. anethifolia, a halophyte plant, which grows in the high-salinity regions in the north of China, where the air temperature in the summer is often as high as 45°C.

Keywords

ArtemisiaChlorophyll fluorescenceGas exchangePhotosystem IISalt adaptationThermotolerance

Abbreviations

ABS

Absorption

CS

Optical cross-section

ET

Energy flux for electron transport

DI

Dissipation

Fo and Fm

Minimal and maximal fluorescence in the dark-adapted state

Fo′ and Fm

Minimal and maximal fluorescence in the light-adapted state

Fs

Steady-state chlorophyll fluorescence level in the light-adapted state

Fv/Fm (φPo)

Maximal efficiency of PSII photochemistry

Fv′/Fm

Efficiency of excitation capture by open PSII centers

ΦPSII

Actual PSII efficiency

ψo

Efficiency with which a trapped exciton can move an electron into the electron transport chain further than QA

φEo

Quantum yield of electron transport beyond QA

RC

Reaction center

TR

Energy flux for trapping

Introduction

The decline in growth observed in many plants subjected to excessive salinity is often associated with a decrease in their photosynthetic capacity (Long and Baker 1986). Since photosystem II (PSII) is believed to play a key role in the response of photosynthesis to environmental perturbations (Baker 1991), the effects of salinity stress on PSII have been investigated extensively. Many studies have shown that salt stress inhibits PSII activity (Masojidek and Hall 1992; Belkhodja et al. 1994; Everard et al. 1994).

Halophyte plants are capable of adapting to highly saline soil. However, previous studies on the effects of salt stress on PSII photochemistry were carried out mainly on non-halophyte plants. There are very few reported studies of how high salinity affects the photochemical aspects of PSII, such as energy absorption, utilization, and dissipation of excess energy, in halophytes.

Artemisia anethifolia is an annual herbaceous halophyte plant. It mainly grows in the regions with saline soil, arid and semi-arid regions with high salinity, and the coastal areas with tidal inundation of seawater in north-eastern, north-western and northern China (Zhao 1998). Under saline conditions, A. anethifolia plants grow slowly. The total number of leaves and the extent of leaf succulence increase (Zhao 1998). In contrast to some other halophytes, A. anethifolia plants have no salt glands or salt bladders on their leaves. The major mechanism for adaptation to high salinity in A. anethifolia is its selective K+ uptake over Na+, i.e. preferential exclusion of Na+ by its roots. In addition, the accumulation of toxic Na+ in the vacuoles can be also considered to be one important aspect for acclimation to highly saline conditions (Zhao and Li 1999).

Very recently, our studies have shown that there are no changes in energy absorption, utilization, and dissipation of excess energy of PSII even when A. anethifolia plants are treated with salinity as high as 400 mM NaCl and exposed to high irradiance at midday, suggesting that A. anethifolia shows high resistance to both high salinity and photoinhibition (Lu et al. 2003).

However, A. anethifolia is also subjected to high-temperature stress since the air temperature in the summer in the north regions of China is often as high as 45°C. Since A. anethifolia can grow in the high-salinity regions while simultaneously exposed to high-temperature stress, we propose the hypothesis that growth of A. anethifolia under high-salinity conditions induces an increased ability to protect itself against high-temperature damage.

Chlorophyll fluorescence analysis has been proven to be a sensitive and reliable method for detection and quantification of changes induced in the photosynthetic apparatus by high-temperature stress (Berry and Björkman 1980; Schreiber et al. 1988; Lichtenthaler and Rindele 1988). Recently, Strasser et al. (1995) have developed a method for the analysis of the kinetics of fast fluorescence rise since the measurements can be done with a high resolution of 10 μs. All oxygenic photosynthetic materials investigated so far show a polyphasic fluorescence rise consisting of a sequence of phases, denoted as O, J, I, and P (Strasser et al. 2000). Strasser and Strasser (1995) have also developed a procedure for quantification of the OJIP florescence transients, known as the JIP test. With this test, it is possible to calculate several phenomenological and biophysical expressions of PSII (Strasser and Strasser 1995; Strasser et al. 1999, 2000). The shape of the OJIP fluorescence transient has been found to be very sensitive to environmental stresses, in particular to high temperature (Srivastava et al. 1997, 1998; Strasser 1997; Strasser et al. 1999, 2000). The JIP test is thus a powerful tool for the in vivo investigation of the behavior of PSII function, including the fluxes of absorption, trapping, and electron transport (Strasser et al. 1999, 2000).

The objective of this study was to investigate whether salt adaptation can induce increased resistance of PSII apparatus to high temperature in A. anethifolia. To this end, we investigated the differences in PSII photochemistry between salt-adapted and non-salt-adapted plants in response to heat stress. We also analyzed the possible differences in the damage sites resulting from heat stress between salt-adapted and non-salt-adapted plants by using fluorescence methods.

Materials and methods

Plant material

Seedlings of A. anethifolia L., the seeds of which were kindly provided by Professor Baoshan Wang at Life Science College, Shandong Normal University, were grown outdoors in plastic pots (14 cm in diameter and 13 cm in height) filled with sand and watered daily with half-strength Hoagland nutrient solution. The average temperature for day/night was 28/19°C, the relative humidity was 60–80% and the maximum photosynthetically active radiation was about 1,500 μmol m−2 s−1.

After 8 weeks, the seedlings were subjected to salt treatment. Salt concentrations were stepped up in 50 mM day−1 increments until final concentrations (0, 100, 200, 300, 400 mM) were achieved. NaCl was dissolved in half-strength Hoagland nutrient solution and plants were watered daily to dripping point with approximately 0.5 l of salt solution. All measurements on the youngest and fully expanded leaves were done 3 weeks after final treatment concentrations were reached, when plants had achieved a steady state.

Heat stress treatments

Heat stress was applied on detached leaves in the dark. The leaves were directly placed into the smooth bottom of a small hole (5.5 cm in height × 3 cm in diameter) in a block of brass (8 cm in height × 8 cm in diameter), the temperature of which was regulated by circulation of water from a thermostatted water bath. At the same time, the leaves were pressed directly by a glass block so that water evaporation from the leaf could be prevented and heat equilibrium between the leaves and the brass block could be reached immediately. The hole in the brass was also covered during treatment. The leaves were exposed to different elevated temperatures (30–45°C) for 30 min and their fluorescence characteristics were measured after heated leaves had been kept in the dark and covered with wet cheesecloth at room temperature for 30 min. During fluorescence quenching analysis, the leaves were placed on the wet cheesecloth to avoid water loss from the leaves. No difference was observed in the rate of temperature increase between control and salt-adapted leaves.

In addition, in order to avoid possible water loss from the leaves during analysis of photosynthetic gas exchange, the leaves attached to the shoots that were supplied with water were used for heat treatments in a high-humidity growth chamber at different temperatures (30–45°C) in the dark for 30 min. Then, gas exchange of the leaves attached to the shoots was measured after heated shoots had been kept in the dark at room temperature for 30 min. The shoots were supplied with water both during analysis of gas exchanges and during incubation at room temperature so that the leaves had no water loss during measurements.

Measurements of chlorophyll fluorescence in modulated light

Chlorophyll fluorescence was measured at room temperature (25°C) with a portable fluorometer (PAM-2000; Walz, Germany) after the leaves had been dark-adapted for 30 min. The fluorometer was connected to a leaf-clip holder (2030-B; Walz) with a tri-furcated fiberoptic (2010-F; Walz) and to a computer with data acquisition software (DA-2000; Walz). The experimental protocol of Genty et al. (1989) was basically followed.

The minimal fluorescence level (Fo) with all PSII reaction centres (RCs) open was measured using modulated light, which was sufficiently low (<0.1 μmol m−2 s−1) not to induce any significant variable fluorescence. The maximal fluorescence level (Fm) with all PSII RCs closed was determined by a 0.8-s saturating pulse at 8,000 μmol photons m−2 s−1 in dark-adapted leaves. Then, the leaf was continuously illuminated with white actinic light at an irradiance of 250 μmol photons m−2 s−1. The steady-state value of fluorescence (Fs) was reached after ca. 5 min illumination and was thereafter recorded. A second saturating pulse at 8,000 μmol photons m−2 s−1 was then imposed to determine the maximal fluorescence level in the light-adapted state (Fm′). The actinic light was then removed and the minimal fluorescence level in the light-adapted state (Fo′) was determined by illuminating the leaf with a 3-s pulse of far-red light. All measurements of Fo and Fo′ were performed with the measuring beam set to a frequency of 600 Hz, whereas all measurements of Fm and Fm′ were performed with the measuring beam automatically switching to 20 kHz during the saturating flash.

Using both light and dark fluorescence parameters, we calculated: (1) the maximal efficiency of PSII photochemistry in the dark-adapted state, Fv/Fm=(FmFo)/Fm (Butler and Kitajima 1975); (2) the actual PSII efficiency, ΦPSII=(Fm′−Fs)/Fm′; (3) the efficiency of excitation capture by open PSII centers, Fv′/Fm′=(Fm′−Fo′)/Fm′ (Genty et al. 1989). Fluorescence nomenclature was according to van Kooten and Snel (1990).

Measurements of polyphasic chlorophyll induction transients (OJIP)

The polyphasic fluorescence transients (OJIP) were measured using a Plant Efficiency Analyzer (PEA; Hansatech Instruments, King’s Lynn, Norfolk, UK) according to Strasser et al. (1995). The transients were induced by red light of about 3,000 μmol photons m−2 s−1 provided by an array of six light-emitting diodes (peak 650 nm), which focused on the sample surface to give homogenous illumination over the exposed area of the sample (4 mm in diameter). The fluorescence signals were recorded within a time scan from 10 μs to 1 s with a data-acquisition rate of 105 points per second for the first 2 ms and of 1,000 points per second after 2 ms. The fluorescence signal at 50 μs was considered as a true Fo since the fluorescence yield at this time was shown to be independent of light intensity.

The JIP test

Chlorophyll fluorescence transients (OJIP) were analyzed according to the JIP test (Strasser and Strasser 1995; Strasser et al. 1999, 2000; Krüger et al. 1997; Appenroth et al. 2001), by using the following original data: (a) the fluorescence intensity at 50 μs considered as Fo when all PSII RCs are open; (b) the maximal fluorescence intensity, Fm, assuming that excitation intensity is high enough to close all the RCs of PSII; (c) the fluorescence intensities at 300 μs (K-step) and 2 ms (J-step). The following parameters are used for the quantification of PSII behavior referring to time zero:
  1. 1.

    The specific energy fluxes (per RC) for absorption (ABS/RC), trapping (TRo/RC), electron transport (ETo/RC) and dissipation (DIo/RC).

     
  2. 2.

    The flux ratios or yields, i.e. the maximal quantum yield of primary photochemistry (φPo=TRo/ABS=Fv/Fm), the efficiency with which a trapped exciton can move an electron into the electron transport chain further than QAo=ETo/TRo), and the quantum yield of electron transport (φEo=ETo/ABS).

     
  3. 3.

    The phenomenological energy fluxes (per excited cross-section, CS) for absorption (ABS/CS), trapping (TRo/CS), electron transport (ETo/CS) and dissipation (DIo/CS).

     
  4. 4.

    The amount of active PSII RCs per excited cross-section (RC/CS) and the total number of active RCs per absorption (RC/ABS).

     

In the JIP test, CS stands for the cross-section of the tested sample. The value of the initial fluorescence Fo or the maximal fluorescence Fm has been proposed as a measure (in arbitrary units) of the phenomenological absorption flux ABS/CS (Strasser and Straser 1995). In this study, we observed that Fo and Fm changed significantly during heat stress. We thus used the chlorophyll concentration per area, ABS/CS (Krüger at al. 1997). We found that there were no differences in chlorophyll content per leaf area between non-salt-adapted plants and salt-adapted plants and that there were no changes in chlorophyll content per leaf area during heat stress. Therefore, ABS/CS can be considered to be constant throughout the experiments.

Analysis of photosynthetic gas exchange

Gas-exchange was analyzed using an open system (Ciras-1; PP Systems, Hitchin, UK). Leaf net CO2 assimilation rate and stomatal conductance (Gs) were determined at 360 μl l−1 CO2 concentration, a leaf temperature of 30°C, 80% relative humidity, flow rate 300 ml min−1, and saturation light intensity (1,000 μmol photons m−2 s−1).

Results

After A. anethifolia plants had adapted to high salinity (100–400 mM NaCl), we examined the changes in the series of fluorescence parameters related to PSII photochemistry derived from the analysis of modulated fluorescence and the polyphasic rise fluorescence transients. At normal temperature, i.e. 30°C, no changes were observed in these parameters after adaptation to different salt concentrations. We thus present only the data for non-salt-adapted plants and plants adapted to 400 mM NaCl. These results suggest that there was no change in PSII photochemistry in salt-adapted plants and that A. anethifolia plants are therefore able to adapt to high salinity. In addition, we observed that there were no significant differences in the responses of the fluorescence parameters of dark- or light-adapted leaves in relation to adaptation of PSII photochemistry to high-temperature stress among A. anethifolia plants treated with various NaCl concentrations (100–400 mM). This result suggests that the increased thermotolerance of PSII induced by salt adaptation, as shown below, is independent of the degree of salinity. Therefore, we present only the results for non-salt-adapted plants and the plants adapted to 400 mM NaCl during heat stress.

Figure 1 shows the responses of the maximal efficiency of PSII photochemistry (Fv/Fm), the efficiency of excitation capture by open PSII centers (Fv′/Fm′), and the actual PSII efficiency (ΦPSII) to elevated temperatures. There was a striking difference in the responses to heat stress between non-salt-adapted plants and salt-adapted plants at temperatures higher than 37.5°C. When temperature was increased to 40°C, Fv/Fm, Fv′/Fm′ and ΦPSII decreased significantly in non-salt-adapted plants but still were unchanged in salt-adapted plants. With increasing temperature, salt-adapted plants showed much higher values of Fv/Fm, Fv′/Fm′ and ΦPSII than non-salt-adapted plants, indicating that salt adaptation induced an increase in the resistance of PSII to heat stress.
Fig. 1a–c

Changes in the responses of the maximal efficiency of PSII photochemistry (Fv/Fm), the efficiency of excitation capture by open PSII centers (Fv′/Fm′), and the actual PSII efficiency (φPSII) in leaves of non-salt-adapted (filled symbols) and salt-adapted (open symbols) Artemisia anethifolia plants following exposure of leaves to elevated temperatures in the dark for 30 min. Each value is mean ± SE of 4 replicates

Figure 2 shows the changes in Fo, Fo′, Fm and Fm′ in response to heat stress. Similar to the change in patterns of Fv/Fm, Fv′/Fm′ and ΦPSII, there was also a striking difference in the responses of Fo, Fo′ and Fm to heat stress between non-salt-adapted and salt-adapted plants at temperatures higher than 37.5°C. At 40°C, Fm decreased while Fo and Fo′ increased significantly in non-salt-adapted plants but they were still unchanged in salt-adapted plants. On the other hand, Fm′ decreased slightly in non-salt-adapted plants only at 45°C but was largely unchanged in salt-adapted plants.
Fig. 2a–d

Changes in the minimal (Fo, a) and the maximal chlorophyll fluorescence (Fm, c) in the dark-adapted state, and in the minimal (Fo′, b) and the maximal chlorophyll fluorescence (Fm′, d) in the light-adapted state in leaves of non-salt-adapted (filled symbols) and salt-adapted (open symbols) A. anethifolia plants following exposure of leaves to elevated temperatures in the dark for 30 min. Each value is the mean ± SE of 4 replicates

We further investigated whether the differences in fluorescence parameters related to PSII photochemistry between non-salt-adapted and salt-adapted leaves following high-temperature stress persisted during recovery from high-temperature stress. We observed that the differences in Fv/Fm, Fv′/Fm′ and ΦPSII between non-salt-adapted and salt-adapted leaves persisted following a 12-h recovery period (Fig. 3).
Fig. 3a–f

Maximal efficiency of PSII photochemistry (Fv/Fm), the efficiency of excitation capture by open PSII centers (Fv′/Fm′) and the actual PSII efficiency (ΦPSII) of non-salt-adapted (open columns) and salt-adapted (filled columns) A anethifolia plants heated at 30 or 45°C for 30 min. a,c,e Leaves immediately following heat treatment; b,d,f the same leaves following 12 h recovery at room temperature. Each value ist the mean and SE of leaves from four separate plants

Figure 4 shows the time courses of Fv/Fm, Fv′/Fm′ and ΦPSII in non-salt-adapted and salt-adapted leaves when exposed to 45°C. The differences in Fv/Fm, Fv′/Fm′ and ΦPSII between non-salt-adapted and salt-adapted leaves were apparent within 5 min of the onset of heat stress.
Fig. 4a–c

Time courses of the maximal efficiency of PSII photochemistry (Fv/Fm, a), the efficiency of excitation capture by open PSII centers (Fv′/Fm′, b) and the actual PSII efficiency (ΦPSII, c) in leaves of non-salt-adapted (filled circles) and salt-adapted (open circles) A. anethifolia plants heated at 45°C. Each value is the mean ± SE of 4 replicates

Figure 5 demonstrates the changes in the efficiency with which a trapped exciton can move an electron into the electron transport chain further than QAo), the quantum yield of electron transport beyond QAEo), and the amount of active reaction centers per excited cross-section (RC/CS) in non-salt-adapted and salt-adapted plants during heat stress. In non-salt-adapted plants, ψo and φEo had already decreased significantly when temperature was increased to 37.5°C. In salt-adapted plants, however, ψo and φEo were largely unchanged at 37.5°C and only started to decrease significantly at 40°C. RC/CS had already started to decrease significantly at 35°C in non-salt-adapted plants but only decreased significantly at 42.5°C in salt-adapted plants.
Fig. 5a–c

Changes in the efficiency with which a trapped exciton can move an electron into the electron transport chain further than QAo, a), the quantum yield of electron transport beyond QAEo, b) and the amount of active PSII reaction centers per excited cross-section (RC/CS, c) in leaves of non-salt-adapted (filled circles) and salt-adapted (open circles) A. anethifolia plants following exposure of leaves to elevated temperatures in the dark for 30 min. Each value is the mean ± SE of 4 replicates

Figure 6 shows the changes in ABS/RC, TRo/RC, and ETo/RC in non-salt-adapted and salt-adapted plants during heat stress. ABS/RC, TRo/RC, and ETo/RC had already increased significantly at 35°C in non-salt-adapted plants but only increased significantly at 42.5°C in salt-adapted plants. With further increasing temperature, these increases were greater in non-salt-adapted plants than in salt-adapted plants.
Fig. 6a–c

Changes in the specific energy fluxes for absorption, trapping and electron transport per PSII reaction center (RC), i.e. ABS/RC (a), TRo/RC (b), ETo/RC (c) in leaves of non-salt-adapted (filled circles) and salt-adapted (open circles) A. anethifolia plants following exposure of leaves to elevated temperatures in the dark for 30 min. Each value is the mean ± SE of 4 replicates

The increased thermotolerance of PSII induced by salt adaptation can be visualized by the energy pipeline model of photosynthetic apparatus (Krüger et al. 1997; Srivastava et al. 1998, 1999; Appenroth et al. 2001). Figure 7 shows the leaf model that refers to the cross-section of leaf and deals with the phenomenological fluxes (per CS). This is a dynamic model in which the energy fluxes in non-salt-adapted and salt-adapted plants as affected by heat stress are expressed by the width of the corresponding arrows. The model also shows the changes in the active (open circles) and inactive (closed circles) PSII reaction centers per cross-section (RC/CS), as well as the flux of dissipated excitation energy at time zero (DIo).
Fig. 7

Energy pipeline model of phenomenological (leaf model, per CS) fluxes in non-salt-adapted and salt-adapted A. anethifolia plants following exposure of leaves to 45°C in the dark for 30 min. The value of each of the parameters can be seen in the relative changes in the width of each arrow. Active RCs are shown as open circles and inactive RCs as closed circles

The most striking effects seen from the leaf model are the greater decrease in the number of active RCs (RC/CS) and the greater increase in energy dissipation (DIo/CS) in non-salt-adapted plants than in salt-adapted plants when exposed to high temperature. The effects are also evident from the greater decrease in trapping (TRo/CS) and electron transport (ETo/CS) on a leaf area basis in non-salt-adapted plants than in salt-adapted plants.

It has been shown that the oxygen-evolving complex is the part of the PSII apparatus most susceptible to heat stress (Berry and Björkman 1980; Havaux 1993a; Lu and Zhang 2000). We further examined whether enhanced tolerance of PSII to high temperature in salt-adapted plants was associated with increased resistance of the oxygen-evolving complex to heat stress.

Non-salt-adapted plants show a typical polyphasic rise of fluorescence transients, including phases O, J, I and P (Fig. 8a). Heat stress can induce a rapid rise in the polyphasic fluorescence transients. The level at around 300 μs has been designated as K, and the O–K transient is the fastest phase observed in the OJIP transient, which, in consequence, becomes an OKJIP transient (Srivastava et al. 1997). It has also been shown that phase K is caused by an inhibition of electron donation from water to the secondary electron donor of PSII, YZ, which is due to uncoupling of the oxygen-evolving system from the Mn-complex. As a consequence, phase K can be used as a specific indicator of damage to the oxygen-evolving complex (Strasser 1997).
Fig. 8 a

Effects of high temperature on the polyphasic chlorophyll fluorescence transients plotted on a logarithmic time scale in control and salt-adapted leaves of A. anethifolia plants. The curves are: control leaves (open circles), salt-adapted leaves (filled circles), control leaves exposed to 45°C for 30 min (open triangles), salt-adapted leaves exposed to 45°C (filled triangles). The relative variable fluorescence at any time t is defined as: Vt=(FtFo)/(FmFo). b Changes in the amplitude of the K-step, which is expressed as the ratio WK=VK/VJ in control (0 mM NaCl, filled circles) and salt-adapted (400 mM NaCl, open triangles) leaves after exposure to elevated temperatures in the dark for 30 min. The insert shows the variable fluorescence intensity normalized to the J-step as W=(FtFo)/(FJFo). Each value is the mean ± SE of 4 replicates

After exposure to 45°C for 30 min, a very clear K-step appeared in non-salt-adapted plants while this heat-induced K-step did not become pronounced in salt-adapted plants (Fig. 8a). In order to compare the changes in the amplitude of the K-step during heat stress, the fluorescence curves were normalized between Fo and FJ, i.e. W=(FFo)/(FJFo) (the insert in Fig. 8b). The amplitude of the K-step (WK) was expressed as the ratio VK/VJ=WK. A higher WK was observed in non-salt-adapted plants than in salt-adapted plants over the range 40–45°C, indicating that salt-adaptation can increase the resistance of the oxygen-evolving complex to heat stress.

We further investigated whether salt adaptation also induced an increase in the resistance of CO2 assimilation capacity to high-temperature stress. Figure 9 shows the changes in net CO2 assimilation rate and stomatal conductance after heat stress in non-salt-adapted and salt-adapted plants. After exposure to 30°C, CO2 assimilation rate and stomatal conductance decreased with increasing salt concentration. After pretreatment at 35°C, CO2 assimilation rate increased slightly in non-salt-adapted plants but significantly in salt-adapted plants. Stomatal conductance increased considerably in both non-salt-adapted and salt-adapted plants. After pretreatment at 42.5°C, non-salt-adapted plants showed a significant decrease in CO2 assimilation rate. However, CO2 assimilation rate in salt-adapted plants was either maintained or even increased to some extent. This greater decrease in non-salt-adapted leaves was not due to the decrease in stomatal conductance since stomatal conductance actually increased in both non-salt-adapted and salt-adapted plants. After exposure to 45°C, net CO2 assimilation rate was below zero in both non-salt-adapted and salt-adapted plants. These results suggest that there was an increase in the resistance of CO2 assimilation capacity to high temperature stress in salt-adapted plants.
Fig. 9a,b

Changes in CO2 assimilation rate (a) and stomatal conductance (b) in leaves of non-salt-adapted (zero NaCl) and salt-adapted (100, 200, 400 mM NaCl) A. anethifolia plants following exposure to elevated temperatures in the dark for 30 min. Bars represent mean ± SE of leaves from four separate plants

In order to determine the sensitivity and dynamic ranges of the fluorescence parameters in non-salt-treated and salt-treated plants in response to heat stress, we compared the different fluorescence parameters in the two sets of plants at different temperatures. For each fluorescence parameter (X) the difference between salt-treated and non-salt-treated plants during heat stress is expressed as a percentage of the value for non-salt-treated plants, i.e. [(Xsalt-treatedXnon-salt-treated)/Xnon-salt-treated]×100. From Table 1, we can see that there is already a difference between non-salt-treated and salt-treated plants for the parameters ABS/RC, TRo/RC, ETo/RC and RC/CS at 35°C, and for the parameters ψo and φEo at 37.5°C, but that a difference does not occur until 40°C for the parameters Fv/Fm, Fv′/Fm′ and ΦPSII. Thus, the parameters ABS/RC, TRo/RC, ETo/RC and RC/CS could be used for early detection of the effects of salt treatments on PSII, with a tremendous gain of information within this critical 5°C.
Table 1

Comparison of fluorescence parameters of salt-treated and non-salt-treated Artemisia anethifolia plants at different temperatures. The values for each parameter (X) are expressed as the relative difference between salt-treated plants and non-salt-treated plants, i.e. [(Xsalt-treatedXnon-salt-treated)/Xnon-salt-treated]×100. The values are the mean ± SE of 4–6 replicates

Fluorescence parameters

[(Xsalt-treated−Xnon-salt-treated)/Xnon-salt-treated]×100

35°C

37.5°C

40°C

Fv/Fm

0

0

+10±0.2

Fv′/Fm

0

0

+11±0.4

ΦPSII

0

0

+19±0.3

ψo

0

+8±0.3

+14±0.8

φEo

0

+9±0.2

+23±0.9

ABS/RC

−25±2.2

−33±2.5

−45±3.3

TRo/RC

−83±4.5

−131±5.2

−204±7.2

ETo/RC

−10±1.2

−18±2.3

−25±2.4

RC/CS

+10±0.5

+21±2.2

+35±3.1

Discussion

Acquisition of PSII thermotolerance in salt-adapted plants

In the present study, our results show that A. anethifolia plants become more resistant to high-temperature damage when grown in high-salinity conditions. This can be seen by the significantly enhanced resistance of PSII, as reflected in the changes in the various fluorescence parameters related to PSII function (Figs. 1, 2, 3, 4, 5, 6).

Our results also show that the resistance of CO2 assimilation capacity to high temperature was enhanced in salt-adapted plants (Fig. 9). Interestingly, we observed that CO2 assimilation capacity was either maintained or increased in salt-adapted plants but decreased significantly in non-salt-adapted plants after exposure to 42.5°C for 30 min, although PSII photochemistry was already significantly inhibited in salt-adapted plants at this temperature. These results suggest that at high temperatures PSII electron transport in salt-adapted plants might not be a limiting factor for CO2 assimilation capacity even when PSII photochemistry is inhibited and that possibly processes other than PSII photochemistry are responsible for increased CO2 assimilation rate in salt-adapted plants at high temperature. At normal temperature, the decrease in CO2 assimilation rate in salt-adapted plants was accompanied by a decrease in stomatal conductance, indicating that the former may have been caused by the latter. At high temperatures, stomatal conductance increased significantly and the CO2 assimilation rate was maintained in salt-adapted plants (Fig. 9b). It is suggested that, in addition to increased thermotolerance of PSII, the increased stomatal conductance in salt-adapted plants may contribute to maintenance of the CO2 assimilation rate under high temperatures. However, the CO2 assimilation rate was significantly inhibited in non-salt-adapted plants under high temperatures. The increase in stomatal conductance in non-salt-adapted plants under high temperatures suggests that the inhibited CO2 assimilation rate in these plants was the result of significantly inhibited PSII photochemistry and possibly the inhibition of Rubisco activation (Salvucci and Crafts-Brandner 2004).

As a result of many studies, PSII is often considered the most heat-sensitive component of the photosynthetic apparatus, even though several studies have shown that electron transport is unaffected at temperatures that inhibit CO2 fixation (Weis 1981a, 1981b). Recent studies support the idea that PSII electron transport is not limiting at temperatures that inhibit CO2 fixation and that CO2 fixation is most sensitive to heat stress due to the inhibition of Rubisco activation via a direct effect on Rubisco activase (Feller et al. 1998; Salvucci and Crafts-Brandner 2004). However, our current study of A. anethifolia shows that PSII is more sensitive than CO2 fixation to a temperature increase up to 42.5°C, which is not in agreement with the above view. In this study, CO2 fixation and PSII photochemistry were not measured immediately after heat treatment of the leaves; instead, they were measured after the heated leaves had been kept at room temperature for 30 min. It has been shown that the inhibition of Rubisco activation by moderately elevated temperatures up to 40°C is fully reversible after the heated leaves are incubated at 22.5°C for 15 min (Feller et al. 1998; Salvucci and Crafts-Brandner 2004). Thus, one possible explanation for our results is that Rubisco activation, and thereby CO2 fixation, had fully recovered after incubation of heated leaves at room temperature for 30 min. However, it should be pointed out that when the temperature was increased to 45°C, CO2 fixation was already below zero in both salt-adapted and non-salt-adapted plants while PSII activity, such as Fv/Fm, was still relatively high in both sets of plants (Figs. 1, 9), suggesting that CO2 fixation was more sensitive than PSII at relatively high temperatures, as shown in this study. This again would support the view that CO2 fixation is the parameter most sensitive to heat stress (Salvucci and Crafts-Brandner 2004).

The conventional fluorescence parameters Fv/Fm, ΦPSII and Fv′/Fm′ are often used to assess PSII function, especially in response to environmental stresses (Schreiber et al. 1988; Lichtenthaler and Rindele 1988). Our results demonstrate that Fv/Fm, ΦPSII and Fv′/Fm′ showed a significant difference between non-salt-adapted and salt-adapted plants only at 40°C or above (Fig. 1). However, the fluorescence parameters derived from the JIP test, ABS/RC, TRo/RC, ETo/RC and RC/CS, already showed a significant difference between non-salt-adapted and salt-adapted plants at 35°C (Figs. 5, 6).

Why did the parameters derived from the JIP test show a greater sensitivity than the conventional fluorescence parameters such as Fv/Fm, ΦPSII and Fv′/Fm′? The ratio Fv/Fm [maximal variable fluorescence (Fv=FmFo)/maximal fluorescence (Fm)] represents an average value of the efficiency of all the PSII units in the measured excited cross-section, i.e. it includes not only the units with activated RCs but also the units with inactivated RCs (those with zero efficiency). Thus, Fv/Fm gives no direct information on the heterogeneity of PSII RCs. On the other hand, several studies using the JIP test have demonstrated that this way of analyzing the fluorescence transients provides access to the trapping flux of the still-active PSII units. Moreover, since the concentrations of the active RCs can be calculated by the JIP test, it is possible to detect whether some of the RCs are inactivated and to estimate this fraction. This is accomplished by using the initial slope Mo=(dV/dt)o and the relative variable fluorescence VJ at the J-step of the OJIP fluorescence rise, which leads to the calculation of the trapping flux (TR) per active RC when the re-oxidation of QA to QA is inhibited (TRo/RC=Mo/VJ) (see Strasser and Strasser 1995; Strasser et al. 1999, 2000; Krüger et al. 1997; Appenroth et al. 2001). It must be stressed that TRo/RC expresses the maximal rate of the closure of RCs as a fractional expression over the total number of RCs that can be closed. This is because it is possible under stress conditions that some RCs are inactivated in the sense of being transformed to quenching sinks without reducing QA to QA reducing centers. In such a case TRo/RC refers only to the active (QA to QA reducing) centers. The same is valid also for two other specific fluxes ABS/RC and ETo/RC, since their derivation is based on the expression for TRo/RC. Thus, an inactivation of PSII RCs will result in an increase in TRo/RC and thereby ABS/RC and ETo/RC. However, it is possible that an increase in TRo/RC may be due to a decrease in the re-oxidation of QA especially after heat treatments with damaging effects. In this study, we observed that Fv/Fm was unchanged but TRo/RC, ABS/RC, and ETo/RC had already increased significantly by 35°C (Figs. 1, 6). We also observed a decrease in the number of active PSII RCs (Fig. 5). According to the JIP test, Fv/Fm can be deconvoluted into TRo/RC and RC/ABS [Fv/FmPo=TRo/ABS=(TRo/RC)×(RC/ABS)], thus we can deduce that the unchanged Fv/Fm was the overall result of the decrease in active RCs which leads to a decrease in the active PSII RCs per total absorption (ABS/RC) and a simultaneous increase in the trapping flux of the still-active RCs (TRo/RC). Thus, the JIP test can detect more sensitively the possible changes in PSII primary photochemistry than the parameter Fv/Fm. In terms of ΦPSII and Fv′/Fm′, they both are regulated not only by PSII photochemistry but also by CO2 assimilation (Demmig-Adams and Adams 1992; Schreiber et al. 1994). The decrease in Fv/Fm will result in a decrease in ΦPSII and Fv′/Fm′. However, any environmental stresses inhibiting CO2 assimilation will also inevitably lead to the down-regulation of both ΦPSII and Fv′/Fm′ via thermal dissipation even though Fv/Fm is not affected, i.e. PSII primary photochemistry is not affected. Therefore, the changes in ΦPSII and Fv′/Fm′ may not always reflect the changes in PSII primary photochemistry.

Since the different behaviors of the two types of treatment were already apparent at 35°C for the fluorescence parameters that take into account the intermediate step FJ between Fo and Fm and the initial slope to the fluorescence rise, the JIP test exhibits more sensitivity in the detection of PSII function than the conventional fluorescence quenching analysis. These results suggest that the parameters derived from the JIP test showed a greater sensitivity than the conventional quenching analysis for the detection of heat stress and that the JIP test can be used as an early stress detector to distinguish the differences between salt-adapted and non-salt-adapted plants in response to heat stress.

Salt adaptation induced different behaviors of PSII in response to heat stress

In order to gain more insight into the enhanced resistance of PSII to heat stress induced by salt adaptation, the structure and function of PSII were evaluated according to the JIP test by measuring the fast chlorophyll fluorescence transients, which can be used as a tool to detect and estimate the PSII behavior (Krüger et al. 1997; Srivastava et al. 1997, 1998, 1999; Strasser et al. 2000). Our results clearly show that one of major events induced by heat stress was the inactivation of PSII RCs. This can be reflected by the decrease in the number of active RCs (RC/CS) at higher temperatures (Fig. 5c). The leaf model provides a visualization of the inactivation of the RCs and the membrane clearly shows an increase in ABS/RC, the average antenna size expressed as absorbing chlorophylls per fully active RC (Fig. 7). However, more importantly, we observed that the decrease in the number of active RCs was much greater in non-salt-adapted plants than that in salt-adapted plants (Figs. 5, 7), suggesting that increased thermotolerance of PSII induced by salt adaptation was associated with an improvement in the thermostability of the RCs.

The oxygen-evolving complex within the photosynthetic apparatus is very sensitive to heat stress (Berry and Björkman 1980; Havaux 1993a; Lu and Zhang 2000). Obviously, an increase in resistance of the oxygen-evolving complex would help to increase the thermoresistance of PSII. In this study of A. anethifolia, we took advantage of the appearance of a K-step in the OJIP polyphasic fluorescence transient to examine if enhanced thermotolerance of PSII in salt-adapted plants was involved in an increase in thermotolerance of the oxygen-evolving complex. Although it is possible that the K-level is equivalent to the J-level, which appears at a somewhat earlier time, it has been shown that the K-step under heat stress is solely related to damage to the oxygen-evolving complex of PSII (Srivastava et al. 1997; Strasser 1997). Therefore, the K-step can be used as a specific indicator of injury to the oxygen-evolving complex on a practical basis. Our results demonstrate that increased thermotolerance of PSII induced by salt adaptation was also associated with an improvement in thermostability of the oxygen-evolving complex, as shown by the appearance of a phase K in the polyphasic fluorescence transients in non-salt-adapted plants (Fig. 8).

Heat damage has been often evaluated by the increase in Fo (Schreiber and Berry 1977; Bilger et al. 1987). When the photosynthetic apparatus is irreversibly damaged by heat stress, Fo increases considerably (Bilger et al. 1987). An increase in Fo induced by heat stress is believed to be due to the physical separation of the light-harvesting complex from the PSII core complex (Schreiber and Armond 1978; Armond et al. 1980; Srivastava et al. 1997). A sharp increase in Fo in non-salt-adapted plants but only a slight increase in Fo in salt-adapted plants at high temperatures (Fig. 2) indicates that salt adaptation might lead to increased thermostability of the light-harvesting complex, thus protecting the physical separation of the light-harvesting complex from the PSII core complex. Since it has been shown that the light-harvesting complex plays a role in the thermostability of PSII (Havaux 1993b), increased thermostability of the light-harvesting complex in salt-adapted plants may help to maintain PSII function on their exposure to heat stress.

It should be pointed out that the temperatures at which RC/CS, WK and Fo started to change significantly were 35, 37.5 and 40°C, respectively, in non-salt-adapted plants. These results suggest that heat stress first induced an inactivation of active PSII centers, and then inactivated the oxygen-evolving complexes and finally resulted in a separation of the antenna system from the PSII centers.

In addition, we found that the temperature at which ABS/RC, TRo/RC, ETo/RC and RC/CS started to change significantly was 35°C in non-salt-adapted plants but 40°C in salt-adapted plants (Table 1, Figs. 5, 6). The measured enhancement of PSII thermotolerance induced by salt adaptation was +5°C. Considering the very sharp temperature-dependence of PSII photochemistry and that the loss of PSII function occurs in a very narrow range of temperature (Havaux 1993b), as well as the fact that PSII function is one of the most heat-sensitive photosynthetic parameters, such an increase in the thermotolerance of the photosynthetic machinery in salt-adapted leaves is of great physiological significance because A. anethifolia plants grow in regions with highly saline soil and simultaneously suffer high-temperature stress during their growing season.

Acknowledgments

The authors thank the referees for their constructive comments in revising the manuscript and Dr. R Rodriguez for providing the Biolyzer software for the analysis of the JIP test. This study was supported by the Frontier Project of the Knowledge Innovation Engineering of the Chinese Academy of Sciences (KSCXZ-SW-326) and support from the Program of 100 Distinguished Young Scientists of Chinese Academy of Sciences to Congming Lu.

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© Springer-Verlag 2004