ACTA PHYSIOLOGIAE PLANTARUM Vol. 28. No. 5. 2006: 433-443
Differences in the physiological state between triticale and maize plants during drought stress and followed rehydration expressed by the leaf gas exchange and spectrofluorimetric methods Tomasz Hura, Stanis³aw Grzesiak, Katarzyna Hura*, Maciej Grzesiak and Andrzej Rzepka** The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Kraków, Poland * Department of Plant Physiology, Faculty of Agriculture and Economics, Agricultural University of Cracow, Pod³u¿na 3, 30-239 Kraków, Poland ** Department of Plant Physiology, Pedagogical Academy, 2 Podbrzezie, 30-054 Kraków, Poland Corresponding author:
[email protected]
Key words: blue and green fluorescence, drought, maize, photosynthesis, red fluorescence, triticale, water potential
rehydration. Increase in the intensity of blue and green fluorescence in maize seems to be the effect of the photoprotection mechanism which prevents damage to PS II through utilisation of excess energy.
Abstract
List of abbreviations: E – transpiration rate, gs – stomatal conductance, LHC – light harvesting complex, PN – net photosynthesis, PPFD - photosynthetic photon flux density, PS II – photosystem II, PS I – photosystem I, Yw – leaf water potential
The studies were carried out in order to estimate differences in the physiological state between triticale and maize plants subjected to drought stress followed by rehydration. The physiological state of the plants was evaluated by measurements of leaf water potential, net photosynthesis, transpiration and stomatal conductance. Spectrofluorimetric methods for the study of blue, green and red fluorescence were applied. We observed that the soil drought induced a greater water loss in triticale leaves than in maize and consequently caused greater injuries to the photosynthetic apparatus. Moreover, triticale plant recovery was slower than in maize plants during the rehydration phase. The effect was probably connected with the higher functional and structural disorganisation of the photosynthetic apparatus observed during drought stress in triticale. Water stress is responsible for damages to photosystem PS II. The worst light utilisation in photosynthetic light conversion was recorded as an increase in the intensity of red fluorescence. Drought stress induced a strong increase in the intensity of blue and green fluorescence in the studied species and it was still high in maize plants during the first day of
Introduction
In recent years, techniques based on the fluorescence phenomenon have become ubiquitous in plant physiology studies. To obtain a full view of plant response to their environment, fluorescence methods are combined with other techniques, in particular, with gas exchange measurements. Among fluorescence techniques the spectrofluorimetric methods are used to study blue, green and red fluorescence emitted by plant leaves (Schweiger et al. 1996, Cerovic et al. 2002). Measurements
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of blue, green and red fluorescence can be used to detect, follow and define the physiological state of plants under drought stress. Blue and green fluorescence originates from plant phenolics, primarily from ferulic acid covalently bound to carbohydrates of epidermal cell walls (Morales et al. 1994, Schweiger et al. 1996, Cerovic et al. 2002, Meyer et al. 2003, Morales et al. 2005).
It was showed that water deficit in maize leaves led to an increase in the intensity of blue and green fluorescence. During rehydration, after drought completion, the restoration of the intensity of blue and green fluorescence close to the control level was observed (Hura 1999). Phenolic compounds may function as a filter for leaf mesophyll and have the capacity to change properties of light falling on leaf through its absorption and transformation to blue and/or green fluorescence. Plant phenolics in the epidermal layer of leaves absorb about 90 % of UV radiation and thus can protect the photosynthetic apparatus (Caldwell et al. 1983). Drought stress predisposes plants to injury of the photosynthetic apparatus through co-acting with UV or visible radiation and the defence mechanism against such radiation may depend on the accumulation of plant phenolics in leaf tissue (Caldwell et al. 1983, Bilger et al. 2001, Schmitz-Hoerner and Weissenbock 2003).
T he
source of red flu o res cence is the protein-bound chlorophyll a of the mesophyll cells (Schweiger et al. 1996, Buschmann and Lichtenthaler 1998). The measurement of red fluorescence is a sensitive and rapid method of recognising stress effects on plants before visible damages occur. The study of light-induced red fluorescence of plant leaves provides basic information of the function of photosynthetic apparatus and performance of photosynthesis. Various stressful environmental factors reduce photosynthesis of growing plants due to their influence on any one or more events associated with photosynthetic process (Dubey 1997).
It was shown that water deficit inhibits photosynthesis not only as a result of stomatal closure, which limits CO2 diffusion to chloroplasts. When drought is prolonged photosynthesis is reduced due to structural and functional changes occurring in the
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photosynthetic apparatus. Drought limits carbon metabolism and utilisation of light phase products, as a consequence, the large amount of harvested light energy, which is harmful to PS II, cannot be converted to chemical energy (Matorin et al. 1982, Kicheva et. al 1994, Cornic and Masacci 1996, Muller and Whitsitt 1996). The increase in the intensity of red fluorescence at the cost of photosynthetic light conversion is often connected with damages of PS II, PS I and LHC under stress conditions (Schweiger et al. 1996, Buschmann and Lichtenthaler 1998, Hura 1999, Buschmann et al. 2000). Limitations of photosynthesis by stomatal as well as non-stomatal mechanisms depend not only on the duration and intensity of drought stress but also on plant species, stages of plant development and age of leaf (Kicheva et al. 1994).
The aim of the presented investigations was to find differences in the physiological state between triticale and maize plants during drought stress and followed rehydration to provide information on plant abilities to recover. This process is pivotal for the maintenance of regular physiological functions in plants. We hypothesized that there would be differences in measured parameters for C3 (triticale) and C4 (maize) species in the course of drought treatment and rehydration. Special emphasis was put on the changes in the measured parameters during recovery as the rapid uptake of water during rehydration has the potential to better elicit the physiological state of plants after drought stress.
Materials and Methods Plant material and plant growth conditions
Plants of spring triticale (x Triticosecale Wittmack) and maize (Zea mays L.) were grown in an air-conditioned greenhouse chamber. Plants were grown at a temperature 23/18 °C (± 2°C) day/night, a photoperiod of 16 h light/8 h dark, 60 ± 5 % relative air humidity (RH) and with a photosynthetic photon flux density (PPFD) of 350 µmol·m-2·s-1. Plants were grown in Mitscherlich pots filled with mixture of soil, peat and sand (1:1:3, v/v/v). From the 12th day after sowing plants were maintained well-watered (control conditions: 65 % of field water capacity – FWC) in growth chambers to allow
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them to adjust to the environment before drought treatment was imposed. Subsequently, drought treatment (30 % FWC) was started and applied for 14 days. After this period, for the next 14 days in case of seedlings earlier subjected to drought, well-watering conditions were re-established. The pots were weighted every day, and the amount of water lost through transpiration was refilled to keep the appropriate weight of pots in each treatment. Young and fully developed leaves of triticale and maize were used for all measurements. Water potential
The leaf water potential was measured (in MPa) by the dew point method with a C-52 thermocouple psychrometer chamber and a HR-33T dew point microvoltmeter (Wescor Inc., Logan, UT, USA). Leaf discs (Æ = 0.7 cm) were cut from the middle part of expanded leaves and immediately placed inside the psychrometer chamber and left to balance temperature and water vapour equilibrium for 30 min before measurements. Spectrofluorimetric measurements Fluorescence emission and excitation spectra were measured using a Perkin-Elmer LS 50B spectrofluorometer. Fluorescence emission spectra of red fluorescence were recorded between 650 and 800 nm. The leaves were excited at 450 nm. The spectral slit widths were set at 5 nm (excitation) and 10 nm (emission).
The excitation spectra of blue and green fluorescence were recorded with an excitation wavelength varied from 250 to 400 nm and an emission wavelength set at 420 nm and 520 nm, respectively. The slit widths for excitation and emission monochromators were adjusted to 10 nm. The cut-off filter (390 nm) was applied to study both blue and green fluorescence. Both exitation and emission spectra were recorded at room temperature. Leaf gas exchange measurements
Leaf gas exchange was measured using a portable gas exchange device CI - 301 CO2 (CID; Inc Vancouver, Washington State USA), to measure net photosynthesis (PN), transpiration rate (E) and stomatal conductance (gs) photosynthetic of fully
developed leaves. Gas exchange measurements were taken at the PPFD of 850 µmol·m-2·s-1 and at 25 °C, a flow rate was set to 83 cm3·s-1. Data presentation and statistical analysis
The
results are presented as figures showing changes in measured parameters for both triticale (figures A) and maize plants (figures B). Based on such values the percent of the control was calculated for each measured parameter (figures C) to compare differences in the physiological state between triticale and maize plants during drought and rehydration.
Each value presented in figures is a mean either of 7 parallel replication in the case of leaf water potential measurements or 10 determinations for both fluorescence and leaf gas exchange measurements. Standard error (SE) was calculated and shown in figures.
Results and discussion
In control conditions leaf water potential of both studied species exhibited similar changes and ranged from –0.88 MPa to –1.17 MPa in triticale (Fig. 1A), while in maize (Fig. 1B) from –0.85 MPa to –1.10 MPa. During drought treatment leaf water potential (Yw) in triticale was gradually reduced as compared to fully irrigated plants. In maize plants the first 5 days, when water supply was withheld, leaf water potential decreased slowly by 10 % of the initial value (Fig. 1C). In the second part of drought treatment Yw in maize began to decline rapidly, but at the end of the drought periods Yw it reached higher values than those measured in triticale plants.
Under rehydration Yw increased from low values close to the control level in triticale (94 % of the control), to higher values in maize plants (120 % of the control). Triticale plants recovered more slowly and did not strictly attain the control level in the course of rehydration period. In maize plants water potential increased by nearly 20 % after 14th day of rewatering in comparison with control plants. Obtained data for both drought and rehydration period show greater water stress for triticale than for maize plants.
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25 10
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Fig. 1. Changes in leaf water potential in triticale (A) and maize plants (B) during 14 days of drought treatment and during 14 days of rehydration. Results in figure C expressed as a percentage of control. Means ± SE (n = 7).
Fig. 2. Changes in net photosynthesis in triticale (A) and maize plants (B) during 14 days of drought treatment and during 14 days of rehydration. Results in figure C expressed as a percentage of control. Means ± SE (n = 10).
The results presented on Fig. 2A and 2B illustrate
2.36 to 2.70 mmol [H2O]·m-2·s-1, respectively. Values of stomatal conductance in the same conditions (Fig. 4A) changed for triticale within 97-130 mmol [CO2]·m-2·s-2, whilst in maize (Fig. 4B) within 106-127 mmol [CO2]·m-2·s-1.
changes in photosynthetic rate (PN) for triticale and maize plants during drought and rehydration in regard to control. In control conditions, mean values of PN for triticale were lower than those observed in maize plants, however much smaller differences were obtained in the case of transpiration rate and stomatal conductance. Transpiration for triticale (Fig. 3A) ranged from 2.35 to 3.13 mmol [H2O] ·m-2·s-1, while in maize (Fig. 3B) it ranged from
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During drought stress, triticale and maize plants showed a decrease in leaf gas exchange as compared to the control plants. As a consequence of low water potential a progressive decline in net photosynthesis, transpiration and stomata conductance
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Fig. 3. Changes in transpiration rate in triticale (A) and maize plants (B) during 14 days of drought treatment and during 14 days of rehydration. Results in figure C expressed as a percentage of control. Means ± SE (n = 10).
Fig. 4. Changes in stomata conductance in triticale (A) and maize plants (B) during 14 days of drought treatment and during 14 days of rehydration. Results in figure C expressed as a percentage of control. Means ± SE (n = 10).
in leaf tissues was observed. We found a large reduction of PN in triticale and maize plants to about 40 % of the initial value during 11 and 14 day of drought treatment (Fig. 2C). In both cases net photosynthesis was strongly reduced and in triticale it was more strongly inhibited than in maize plants with the exception of the last day of drought stress.
the case of triticale plants (Fig. 2A). It suggests that changes in photosynthesis caused by dehydration for both triticale and maize plants were fully reversible. Recovery rate of net photosynthesis for maize plants was slow in the first part of rehydration and probably due to disturbances in stomatal opening (Fig. 4B). In the second part the maintenance of well-watered conditions caused a small increase in photosynthetic rates in triticale and much higher in maize plants, above the control level (Fig. 2C).
Rehydration after 14 days allowed the recovery of photosynthetic activity to the control rates within 4 days for maize plants (Fig. 2B) and within 7 days in
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Fig. 5. Examples of fluorescence emission spectra of red fluorescence recorded during the last day of drought treatment (a) and during the first day of rehydration (b) for both triticale (thin lines) and maize plants (thickened lines). Continuous line – control plants, broken line – droughted and rehydrated plants.
Moreover, net photosynthesis, transpiration and stomata conductance in maize plants indicated a higher increase than measured in triticale during the second week of rehydration. These results showed that plants were able to restore the physiological activity when Yw was recovered. Reduction in leaf water potential leads to stomata closure and consequently to the inhibition of photosynthesis (Kramer and Boyer 1995). The resistance of the plants to water deficit may partly result from the maintenance of photosynthetic capacity by the leaves al lowing re cov ery dur ing rehydration (Cornic et al. 1989, Quick et al. 1992). Decrease in CO2 assimilation observed in maize can be attributed to stomatal closure as stomatal opening during the first days of rehydration was followed by an increase in net photosynthetic rate. On the contrary, a quick recovery of stomata conductance to the control level observed in triticale did not affect changes in net photosynthesis during the first week of rehydration. One of the earliest responses to drought stress is stomata closure, which limits CO2 diffusion to chloroplasts. It was shown that at moderate water deficit photosynthesis is inhibited as a result of stomatal closure (Itoh and Kumura 1986, Heitholt et al. 1991). However, when drought is prolonged photosynthesis can be reduced due to struc tural and func tional changes in the photosynthetic apparatus (Kicheva et al. 1994,
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Cornic and Masacci 1996, Muller and Whitsitt 1996). Thus, it can be concluded that drought, probably injured the photosynthetic apparatus of triticale.
Figure
5 shows spectra of red fluorescence obtained for triticale (thin lines) and maize leaves (thickened lines) during the last day of drought stress (a) and during the first day of rehydration (b). The continuous line represents control plants while the broken line represents droughted and rehydrated plants. Fluorescence emission spectra of maize plants showed much higher intensity than those measured in triticale plants.
Water stress triggered an increase in emission of red fluorescence. The intensity of red fluorescence during drought stress for both triticale (Fig. 6A) and maize plants (Fig. 6B) was higher than the control one. Thus the efficiency of light utilisation in the control plants was higher than in droughted plants. During the first week of rehydration differences in intensities of red fluorescence between triticale and maize were observed (Fig. 6C). We recorded a decrease in the intensity of red fluorescence in maize plants from the second week of drought stress and such tendency was continued starting from the first day of rehydration. Additionally, the intensity of red fluorescence in maize plants during recovery was lower than observed in
DIFFERENCES IN THE PHYSIOLOGICAL STATE ...
Fig. 6. Changes in red fluorescence intensity in triticale (A) and maize (B) leaves during 14 days of drought treatment and during 14 days of rehydration. Results in figure C expressed as a percentage of control. Means ± SE (n = 10).
Fig. 7. Changes in blue fluorescence intensity in triticale (A) and maize (B) leaves during 14 days of drought treatment and during 14 days of rehydration. Results in figure C expressed as a percentage of control. Means ± SE (n = 10).
control plants. In triticale under well-watered conditions the intensity of red fluorescence was initially higher, but during the second week of rehydration it decreased and achieved the level characteristic for the control plants. Recorded changes in the intensity of red fluorescence in the course of recovery for both studied species are connected with full recovery of the photosynthetic apparatus. However, in triticale an increase in the intensity of red fluorescence during the first phase of
recovery was still observed. In contrast, maize plants recovered photosynthetic efficiency even during the second part of drought stress due to low emission of red fluorescence. On this basis it can be suggested that injuries to the photosynthetic apparatus at the molecular level had a dominant effect on photosynthesis rate in triticale plants during the second half of drought stress.
Water stress is responsible for damages of PS II, PS I and LHCs (van Rensburg and Krüger 1993, Giardi
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Fig. 8. Changes in green fluorescence intensity in triticale (A) and maize (B) leaves during 14 days of drought treatment and during 14 days of rehydration. Results in figure C expressed as a percentage of control. Means ± SE (n = 10).
et al. 1996, Dubey 1997). The reduction of PS II is related to the increase in the emission of red fluorescence (Lang et al. 1996, Lichtenthaler 1996, Schweiger et al. 1996, Šestãk and Šiffel 1997, Buschmann and Lichtenthaler 1998). Additionally, uncoupling of LHC from the reaction centre and structural disorganisation of LHCs may lead to a decrease in excitation energy transfer and consequently to the emission of red fluorescence from plant leaves (Siegenthaler and Rawyler 1977,
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Fig. 9. Examples of fluorescence excitation spectra of blue fluorescence recorded during the last day of drought treatment (a) and during the first day of rehydration (b) in both triticale (thin lines) and maize plants (thickened lines). Continuous line – control plants, broken line – droughted and rehydrated plants.
Panda et al. 1986, Swain et al. 1990, Behera and Choudhury 1997, Tambusi et al. 2000). Several studies showed that PS II was affected by water stress which resulted in lowering the electron transport (Boyer and Bowen 1970, Govindje et al.1981, Chaves 1991, Cornic et al. 1992, van Rensburg and Krüger 1993, Dubey 1997). Some investigations demonstrated that water stress caused damages to the oxygen evolving complex of PS II (Canaani et
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Fig. 10. Examples of fluorescence excitation spectra of green fluorescence recorded during the last day of drought treatment (a) and during the first day of rehydration (b) in both triticale (thin lines) and maize plants (thickened lines). Continuous line – control plants, broken line – droughted and rehydrated plants.
al. 1986, Toivonen and Vidaver 1988) and PS II reaction centres (Havaux et al. 1986, 1987). However, other studies indicated that PS II could tolerate high levels of water deficit and mild desiccation did not substantially decrease PS II activity (Cornic and Briantais 1991, Havaux 1992).
Distinct differences were obtained for changes of blue and green fluorescence. In control conditions maize leaves after excitation emitted blue (Fig. 7B) radiation of higher intensity than triticale (Fig. 7A). In the case of green fluorescence similar results were observed. The control plants of maize (Fig. 8B) emitted higher intensity of green fluorescence than triticale (Fig. 8A). Drought stress strongly induced an increase in the intensity of blue (Fig. 7C) and green fluorescence (Fig. 8C) from leaves. The yield of blue and green fluorescence during the first day of rehydration was still higher in maize plants.
Figures 9 and 10 show excitation spectra of blue and green fluorescence recorded under drought stress and rehydration for triticale and maize plants, respectively. The maximum excitation wavelength for blue and for green fluorescence was between 320-360 nm. The increase in the intensities of blue and green fluorescence is connected with the accumulation of phenolic compounds in plant leaves, which can function as a light-filter for the photosynthetic apparatus (Hideg et al. 2002, Maheswarn
et al. 1987, Lang et al. 1992, 1994, Lichtentaler et al. 1996, Schweiger et al. 1996). This indicates that plant phenolics have the capacity to stretch the path of light falling on leaves. They can transform the potentially harmful violet and blue radiation into blue and green fluorescence no longer damaging PS II system (Björkman and Powels 1984, Šesták and Šiffel 1997, Buschmann and Lichtenthaler 1998, Lichtentaler and Schweiger 1998).
In conclusion, drought stress induced a higher leaf water loss especially during rehydration in triticale than in maize plants. Measured parameters for triticale plants recovered more slowly in comparison with maize plants, what is connected with struc tural and func tional changes in the photosynthetic apparatus. Increase in the intensity of blue and green fluorescence in maize is the effect of the photoprotection mechanism which prevent damage to PS II through the utilisation of excess energy. This protective mechanism enables maize to come out of light stress already during the drought stress by the removal of photoinhibitory injuries. Triticale cannot synthesise organic compounds efficiently, as maize does, because it exhibits a lower carboxylation activity during drought periods. This less effective photoprotective mechanism in triticale causes greater photoinhibitory injuries to occur and consequently it takes longer for
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triticale to remove injuries to the photosynthetic apparatus as compared with maize.
For further research it seems to be of value to determine whether the drought resistance of crop plants might be connected with the phenolics content especially with the concentration of ferulic acid as the main source of blue-green fluorescence.
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Received November 07, 2005; acceptet September 26, 2006 edited by Z. Starck
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