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Reaction of Photosynthetic Apparatus to Dark Desiccation Sensitively Detected by the Induction of Chlorophyll Fluorescence Quenching
JOUR.ALOF.
j. Plant Physiol. Vol. 155. pp. 399-406 (1999) http://www.urbanfischer.de/journals/j pp
PIani PbysjllllY © 1999 URBAN & FISCHER
Reaction of Photosynthetic Apparatus to Dark Desiccation Sensitively Detected by the Induction of Chlorophyll Fluorescence Quenching 1 1 MARTINA MATOUSKOVA , HANA BARTOSKOVA , JAN NAUSl,
and
RADKO NOVOTNy.2
1
Department of Experimental Physics, Palacky University, ti'. Svobody 26, 77146 Olomouc, Czech Republic
2
Center for Microscopic Methods, Faculty of Medicine, Palacky University, 1. P. Pavlova 35, 77520 Olomouc, Czech Republic
Received September 14, 1998 . Accepted February 2, 1999
Summary
Chlorophyll fluorescence parameters Fo, FM, Fy/FM' Fy/Fo, the emission and excitation spectra and induction of fluorescence quenching coefficients qP and qN were followed during the desiccation of leaf segments of spring barley (Hordeum vulgare L. cv. Akcent) and detached leaves of an endohydric moss (Rhizomnium punctatum Hedw.) exposed to the same dark-desiccation conditions (35 % air relative humidity, 24°C). A sensitive fluorescence parameter in the initial phases of tissue desiccation was looked for. A decrease in relative water content (RWC) was much faster in the moss leaves (to 30 % RWC in 10 min) than in barley leaves (to about 30% RWC in 17h). A rather insensitive parameter, Fy/F M, started to decrease at 60 % RWC for the moss and at 30 % RWC for the barley. A more pronounced decrease in comparison with Fy/FM was observed in the case of the Fy/Fo. The ratio of bands (F 685/F 735) in the emission spectra at two excitation wavelengths (436 and 475 nm) was significantly changed only in desiccating R. punctatum. However, the decline in the F 685/F 735 ratio could be ascribed mostly to the increased fluorescence reabsorption. The excitation spectra were practically insensitive to the desiccation process in both species. Much more sensitive parameters reflecting the initial decrease in water content in the tissue were the qP and especially qN coefficients detected during induction elicited by the actinic light. When using the actinic irradiance for the induction similar to that of plant cultivation, the optimal interval for qp and qN detection in barley included values measured in 22 and 42 s of induction. The induction time interval of qP and qN sensitivity was much larger for the moss (included values measured in 2, 22, 42 and 62 s). Based on a distinct character of the induction curves and different changes in the Fo and FM parameters, different strategies of the barley and moss leaves in response to the same desiccation conditions were suggested.
Key words: Barley, chlorophyll fluorescence, desiccation, moss, relative water content. Abbreviations: Chi a and b = chlorophyll a and b; a + b and alb = sum and ratio of Chi a and b, respectively; Chi = chlorophyll; Fo = minimal Chi fluorescence intensity of the dark-adapted leaf; FM and FM' = maximal Chi fluorescence intensity of the dark- and light-adapted leaf; Fy/FM = (FM-FO)/FM = maximal quantum efficiency of photochemistry of PSII; LHC = light harvesting complex; PAR = photosynthetically active radiation; PSII = Photosystem II; OA = primary quinone acceptor ofPSII; qp and qN = photochemical and non-photochemical Chi fluorescence quenching coefficients; RWC = relative water content; x + c = total carotenoid content. 0176-1617/99/155/399 $ 12.00/0
400
MARTINA MATOUSKOVA, HANA BARTOSKOVA, JAN NAus, and RADKO NOVOTNY
Introduction
Responses of plants to the shortage of water leading to water stress are very variable and a variety of alterations in photosynthesis can be found (Hsiao, 1973). The extent of the effects of water stress is dependent on the intensity and duration of the stress as well as on the genetically determined capacity of plant species to cope with water deficit. It is thought (Schreiber and Bilger, 1987; Schwab et a!., 1989; Comic et a!., 1992) that in vascular plants the decrease in the rate of photosynthesis in initial phases of water stress is caused mainly by an inhibition of dark-reactions of photosynthesis. This inhibition occurs because of i) a decreasing intercellular CO 2 concentration due to stomatal closure (Kaiser, 1987) and ii) an inhibition of the activity of Calvin cycle enzymes (Schreiber and Bilger, 1987). A direct effect of desiccation on electron transport in photosystem I! (PSII) was observed when severe dehydration had been reached (see Comic et a!., 1992). In bryophytes the stomatal limitation of photosynthesis is impossible because bryophyte gametophytes have no stomata (Peciar et al., 1984). However, the other photosynthetic responses to water stress are similar to those of vascular plants (Tuba et a!., 1996). Measurement of the chlorophyll fluorescence induction curve is a very useful method for detection of changes in the photosynthetic apparatus due to stress constraints (e. g. Lichtenthaler and Rinderle, 1988). One of the parameters most widely studied with plants in general is Fy/F M , the maximal quantum efficiency of PSI! (Krause and Weis, 1991). This parameter has been found to be constant in initial phases of water stress in various types of plants (Havaux, 1992; Seel et a!., 1992; Eickmeier et a!., 1993; Chen and Hsu, 1995). In accordance with the above mentioned desiccation response it can be expected that the ChI fluorescence parameters reflecting electron transport beyond PSI! will be changed during mild dehydration rather than Fy/FM (Genty et a!., 1987; Stuhlfauth et a!., 1988). In this work, we used several ChI fluorescence methods for the study of the photosynthetic res po me of plants exposed to the same dark-desiccation conditions. We looked for a sensitive ChI fluorescence parameter changing in the initial phases of tissue dehydration and intended to compare its sensitivity in plants with different kinetics of water loss. For this reason we have chosen a monocotyledon Hordeum vulgare L. (cv. Akcent) and an endohydric moss Rhizomnium punctatum Hedw., two plants with different kinetics of desiccation. A sensitive time interval of an induction of quenching coefficients was found for the different physiological reactions of these plants.
Materials and Methods Plant material and stress treatment
Plams uf spring barley (Hordeum vulgare L. (v. Akccnt) were grown in a growth chamber at (20 ± 2) "C and relative air humidity (RH) of (60 ± 5) % on artificial soil composed of perlite and Knopp solution. The light regime was 8 h dark/16 h light (fluorescem light, Tesla Z 25W) with PAR intensity of 90 ~mol m 2 s-l The primary
leaves of plants in the growth phase 1.2 (two leaves) according to Feekes (1941) were used for the experiment. Gametophytes and sporophytes of the moss Rhizomnium punctatum (Hedw.) were collected in November 1997 near a small waterfall ReSov near Rymafov, north-east of the Czech Republic (49" 50' N, IT 15' E). Moss tutves together with a 0.5 em layer of soil were placed in covered Petri dishes in a growth chamber and kept under the above mentioned dark/light regime for 10 days. The RH of the air above the plants was 70 % and the cultivation PAR intensity was 10-15 ~mol m- 2 S-I. For sample preparation a 2-cm long segment of a barley leaf blade was cut off 1.5 cm from the tip of the leaf or several whole moss leaves were detached from the gametophytes. The samples were placed into Petri dishes and dried for 40 h (barley) or 1.5 h (moss) in stationary air in the dark. The conditions of the process of desiccation were T = (24 ± 1) "C and RH = (35 ± 1) % (Humidity and Temperature Meter HM 34C, Vaisala, Helsinki, Finland).
Determination of relative water content
The RWC of both barley and moss samples expressed in percentage of water-saturated tissue were calculated using the equation: RWC = ( mfresh-mdry ). 100 ffisaturated - ffidey
The msaturated of the barley segments was determined from the weight minit measured immediately after detachment of the segments by the relation msarurated = f . minit. The filling coefficient f was found by an independent measurement to be 1.03 ± 0.01. The fresh weight (mfresh) was estimated at a given time of desiccation and the dry mass weight (mdr ) after drying at 105 "C for 60 min. In the case of moss leaves the saturated weight (msarurated) was taken to be equal to the minit and the dry mass weight was determined after drying ofleaves at 105"C for 15 min. Light microscopy
Parts of the hydrated H. vulgare and R. punctatum leaves were fixed for 4 h in 3 % glutaraldehyde under vacuum and postfixed for 4 h in 1 % OS04 in 0.1 mol L -I phosphate buffer, pH 7.2. The samples were dehydrated in a graded acetone series with uranylacetate and embedded in Durcupan ACM (Fluka). The 1 ~m sections were mounted on slides and were stained with toluidine blue. The micrographs were made with a light microscope Jenamed Variant (Carl Zeiss Jena, Germany). Chl fluorescence measurements In vivo ChI a fluorescence was measured at room temperature with a modulation fluorometer PAM 2000 (Walz, Effeltrich, Germany). The leaf samples were dark-adapted (for a minimum time of 2.5 h) before the beginning of experiments. The initial Chi fluorescence intensity of the dark-adapted samples (Fo) was obtained upon excitation with a weak measuring beam (0.08 for H vulgare and 0.3 ~mol m- 2 S-I for R. punctatum). The higher intensity in the latter case was chosen to ensure a reliable signal. An 800 ms saturating pulse was used for determination of the maximal fluorescence intensity (F M ) of the dark-adapted samples. The correct intensity and length of the pulse was checked via kinetics of the Chi fluorescence response to the light pulse. Maximum ChI fluorescence intensity during the actinic illumination (F'M) was determined using additional saturating pulses (see Van Kooten and Snell, 1990). The first pulse was applied 2 s after switching on actinic PAR and the following ones in 20 s intervals. The 1 - qP parameter was calculated using the equation 1 - qp = 1 - (F' M- F,l/(F'M-F o), where Ft was the ChI
401
Dark-desiccation and Chi Fluorescence fluorescence intensity at the time t of the actinic illumination. The coefficient of non-photochemical quenching (qN) was calculated from the equation qN = (FM-F'M)/(FM-FO) (Schreiber et al., 1986). A general statistical description (medians and quartiles) was used in the case of the parameters of ChI fluorescence (Lazar and Naus, 1998). The segments of H. vulgare leaves placed in the Walz clips were irradiated by actinic PAR with an intensity of about 70 !lmol m -2 S-I. A leaf of R. punctatum was placed on a plastic pad and covered with a black mask with a round aperture (radius of 2 mm). The moss leaves were irradiated by actinic light of about 20 !lmol m- 1 S-I. The intensities of the actinic PAR were chosen to reach a comparable intensity to that of cultivation. The fast ChI fluorescence induction (the O-J-l-P transient) of the dark-adapted leaf samples was measured using a portable fluorometer PEA (Hansatech, Norfolk, England). The excitation intensity of PAR was 4,300 !lmol m -2 s-I and the exposure time was 2s. ChI fluorescence emission and excitation spectra at 77 K were measured using a Fluorescence Spectrophotometer F-4500 (Hitachi, Tokyo, Japan). The leaf samples were mounted on a holder and immersed into an optical Dewar flask with liquid nitrogen. The emission spectra (in a range from 650 to 800 nm) were measured at the excitation wavelengths of 436 nm (preferentially ChI a excited) and 475 nm (mainly ChI b together with carotenoids excited). The spectral halfWidths of the excitation and emission monochromators were 5 and 2.5 nm, respectively. The corrected excitation spectra (in a range from 400 to 550 nm) were measured at the emission wavelengths of 685 and 735 nm. The spectral halfWidths of the excitation and emission monochromators were 2.5 and 5 nm, respectively.
Pigment determination The contents of ChI a and b and the sum of carotenoids of the leaves were determined in 80 % acetone according to Lichtenthaler (1987a) using a DU-8 UV-VlSIBLE Spectrophotometer (Beckman, USA).
100
A 0
I
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\
~( 10
20
20
40
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20
40
60
80
Time [min]
Fig. 1: Changes in RWC with time for H. vulgare leaf segments (A) and detached leaves of R. punctatum (B) desiccated in the dark at 35 % RH and 24 ·C for 40 h (barley) and 90 min (moss), respectively. Means and SD are presented, n = 7 (barley) or n = 5 (moss).
Table 1: The effect of dark-desiccation on photosynthetic pigment content, pigment ratios, and ratios of bands in the emission and excitation ChI fluorescence spectra (77 K) of R. punctatum and H. vulgare leaves. The time needed to reach 8 % (moss) and 10 % RWC (barley) was 1.5 hand 40 h, respectively. The pigment values are given as mgg- I initial fresh weight. R. pUJ'/ctatum
Parameter
H. vulgare
RWC=IOO%
RWC=8%
RWC=96%
RWC= 10%
2.36±0.51 1.12±0.25 0.66±0.14 2.12±0.06 5.23±O.18
2.18±0.43 1.01±0.17 0.64±0.12 2.16±0.10 4.97±0.23
l.28±0.16 0.40±0.0,) 0.31 ±0.04 3.19±0.06 5.4HO.13
0.87±0.11'" 0.30±0.OS'" 0.29±0.03 2.94±0.22' 4.07±0.75"
1.04±0.21 l.3S±O.34 0.88±O.OS 1.1O±O.03
O.56±O.II" 0.67±O.16" 0.86±O.OS l.O2±O.03'"
0.34±0.01 0.39±O.OI 1.02±0.O3 0.90±0.O2
O.4HO.IS O.41±0.09 l.lHO.15 1.13±0.04'"
Pigment content
Chla (a) Chlb(b) alb
The decrease in relative water content of the barley leaf segments and detached moss leaves desiccating under the same external conditions was characterized by different kinetics (Fig. 1). The RWC decline in the barley segments was gradual within 40 h up to 10 % (Fig. 1 A). The initial water loss (to about 30 % RWC in 17 h) could correspond mostly to the loss of transpiration water and the water from the epidermal cells (Fig. 2 a). In the leaves of R. punctatum the RWC declined steeply during the first 20 min (to 30 % RWC in 10 min) to a value of 13 % (Fig. 1 B) and might be ascribed to the loss of symplastic water from the cells. The initial water loss was about 100 times faster in the moss leaves than in barley segments. A relatively slight following decline (to 8 % of RWC) was recorded during the next 70 min of dehydration and could be ascribed to the loss of apoplastic water from the cell walls (Dilks and Proctor, 1979; Proctor, 1982). Since leaves of R. punctatum are composed mostly of one or two cell layers and possess no epidermis and only a thin cuticle (Fig. 2 b) (Proctor, 1982; Peciar et aI., 1984; Proctor, 1984) the evaporation from all cells was very rapid.
30
40
I
Time[h]
(a+ b)/(x+c)
Kinetics ofthe RWC decrease
l . . . .,
,
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60
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R. punctatum
i,
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Carotcnoids x + c
Results
,
H. vulgare
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Chi fluorescence ratios F68S/F73S (exc. F685/1'735 (exc. E436/E475 (em. E436/E475 (em.
436 475 68') 73')
nm) nm) nm) nm)
Means and SD are shown, n :::: 7. Asterisks indicate statistically significant differences between hydrated and desiccated Ieafsamples (' P
Pigment content and pigment ratio Significant changes in pigment content and pigment ratios due to dehydration were observed only in the case of the barley leaf segments (Table 1). The desiccation of these segments was accompanied by a decrease in the chlorophyll content by about 30 %, whereas the total carotenoid content was maintained. Thus, the (a + b)/(x + c) ratio decreased during desiccation of the barley segments, which represents a similar tendency of the pigment breakdown as during autumnal senescence (Merzlyak and Gitelson, 1995). The slight decrease in the ChI alb ratio indicates that the content of ChI a declined more rapidly than the ChI b content. It should be noted here that the lower Chi alb ratio in hydrated moss leaves in comparison with barley indicates a shade adaptation of the moss plants (Anderson et aI., 1988; Martin and Warner, 1984).
402
MARTINA MATOUSKOVA, HANA BARTOSKOVA, JAN NAus,
and RADKO
b
Fig. 2: The light microscope phorographs of 111m leaf cross-sections of hydrated Hordeum vulgare (a) and Rhizomnium punctatum (b). E, epidermis; M, mesophyll; VB, vascular bundle. Bars 100 11m.
Chi fluorescence parameters Changes in the Fo, FM, Fv/FM and Fv/Fo parameters during desiccation of the moss leaves and barley leaf segments are shown in Fig. 3. The basic Fo and FM quantities are less precise as they reflect individual properties of the samples and may be affected also by the changing leaf structure due to desiccation. However, the changing leaf optical properties should change both quantities in a similar manner. This was not the case (Fig. 3A, B). In the desiccating moss leaves, Fo increased steeply about 2 times while FM remained constant (Fig. 3 A, B). The increase in Fo in the case of R. punctatum deserves more detailed attention. A slightly higher analytical PAR intensity than that of barley was used to ensure a reliable signal. It may be argued that this intensity of PAR might
NOVOTNY
have increased the measured Fo level due to OA reduction in some PSI! centres. Therefore, the Fo dependence was checked by a quite different method of Fo estimation from the very fast fluorescence induction kinetics (the O-J-I-P curve) measured by PEA, Hansatech (Strasser et aI., 1995; Lazar et aI., 1997). The increase in Fo was confirmed (Fig. 3 A dashed line). In contrast to moss, the desiccation of barley leaf segments led to only slight changes in Fo and to a decrease in FM to about 60 % of the initial level (Fig. 3A, B). It has been verified experimentally that the FM decrease in barley was not caused by a mere senescence in darkness (data not shown). Figure 3 C shows that the ratio Fv/FM declined during the desiccation of both H vulgare and R. punctatum. However, the rate and extent of this decline were different. In the case of moss the Fv/FM decline started at 60 % of RWC from the value of 0.71 and decreased to the value of 0.27 for 8 % RWC. The Fv/FM ratio of barley remained constant up to 30 % of RWC and then decreased from a value of 0.79 to a value of 0.59 for 10 % RWC. The value of Fv/FM of the most desiccated samples represented about 40 % (moss) and about 75 % (barley) of the Fv/F M value of the hydrated samples. In the case of the Fv/Fo ratio, a more pronounced decrease in comparison with Fv/FM was observed (Fig. 3 D). The values of Fv/Fo decreased in the most dehydrated samples to 25 % (moss) and 40 % (barley) of the values of the hydrated leaves. The ratio of fluorescence emission bands F685/F735 at two excitation wavelengths (436 and 475 nm) and the ratio of fluorescence excitation bands E436/E475 at two emission bands (F685 and F735) measured at 77K were also followed (Table 1). The most significant change found was the decrease in F685/F735 emission ratio in the case of R. punctatum upon desiccation to 8 % RWC by about 50 %. (The values of the band ratio were measured in 5 points of the dependence and they gradually decreased; only the first and last values are shown in Table 1.) This large change in the F685/F735 ratio without changes in the chlorophyll content could be explained by a strongly enhanced effect of Chi fluorescence reabsorption due to the clustering of chloroplasts in the dehydrated moss cytoplasm (Naus et aI., 1994). On the other hand, H vulgare revealed a tendency to increase this ratio. However, the desiccation of the barley segments was accompanied by the decrease in chlorophyll content by about 30 %. Thus, the reabsorption effect decreased due to a decrease in the chlorophyll concentration (Lichtenthaler, 1987b; Lichtenthaler and Rinderle, 1988; Buschmann and Lichtenthaler, 1998; Gitelson et aI., 1998). Although the effect of Chi fluorescence reabsorption dominates in the emission spectra the excitation spectra are not influenced because the absorbances at 436 and 475 nm are known to be similar (Lee et aI., 1990; Agati et aI., 1993). The changes in the Chi fluorescence excitation spectra were much lower and complicated to be interpreted as a complex combination of the optical, reabsorption and energy transfer changes that might have appeared. However, it seems that changes in energy transfer were not greater than 15 % based on the excitation spectra measurements. Thus, the excitation spectra in Table 1 help to exclude such mechanisms of the Fo increase and F685/F735 decrease in the R. punctatum leaves which are connected with significant changes in the energy transfer.
403
Dark-desiccation and Chi Fluorescence
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Fig. 3: Changes in the Chi fluorescence parameters Fo (A), FM (B), Fy/FM (C) and Fy/Fo (D) as measured with the PAM fluorometer by the application of a 0.8 s saturation pulse on darkadapted H. vulgare leaf segments and R. punctatum leaves during desiccation in the dark at 35 % RH and 24 'c. The values of Fo measured with the PEA instrument (Hansatech) in desiccating R. punctatum leaves are shown (- - - -... - - - -). Medians and quartiles are presented, n = 7. Asterisks indicate statistically significant differences between hydrated (96 % RWC for barley and 100 % RWC for moss) and desiccating leaf samples (* P<0.05; ** P
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The courses of 1 - qP and qN parameters during the first minutes of induction caused by actinic PAR are shown in Fig. 4. To measure the induction of quenching parameters qP and qN, we used the intensity of PAR close to the PAR of cultivation. This was done to avoid photo inhibition under too high intensity and insufficient excitation under too low PAR intensity. During the period of the actinic illumination the hydrated (non-stressed) H. vulgare as well as R. punctatum leaves exhibited characteristic induction kinetics of the 1 - qP and qN parameters (see Schreiber et aI., 1986). As is shown in Fig. 4, the changes in the induction curves due to desiccation were quite different for the barley and moss samples. In the case of barley, the steady-state values of 1- qp and qN differed only slightly upon the decrease in RWC from 96 % to 40 %. When RWC was 13 %, no decline in 1 - qP and only a gradual increase in qN without any relaxation were observed. In the moss leaves of 60 % RWC a decrease in 1 - qP within the first 2 min of illumination was followed by a gradual increase (Fig. 4 B). This effect was probably caused by a continual desiccation of the samples during the fluorescence induction (compare with Fig. 1 B). A similar effect was found in the case of qN in the moss leaves of 80 % and 60 % RWC (Fig. 4 D). When the RWC dropped to 40 %, only a progressive increase in both parameters was observed. The samples with 13 % RWC revealed no functional processes. The induction curves of the Chi fluorescence quenching in moss and barley leaves differed also in another aspect. In the
60
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RWC[%]
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100
80
60
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0
RWC[%]
case of H. vulgare only a tendency of changes in one direction was detectable. The qN coefficient increased in stages of induction near to steady state (3 to 5 min of induction). The qN parameter of R. punctatum increased already at 80 % RWC, reached its maximum for about 60 % RWC and decreased upon further drying. In the case of the moss leaves two phases of the desiccation effect were discernible.
Discussion
Sensitivity of Chi fluorescence parameters It has been reported that the photosynthetic processes related to thylakoid membranes are very resistant to drought conditions (Havaux, 1992; Schwab et aI., 1989) and that a mild water stress causes mainly an inhibition of dark-reactions of photosynthesis (Kaiser, 1987; Schreiber and Bilger, 1987). These conclusions result among others from measurements of chlorophyll fluorescence parameters whose changes started only when water stress became severe. Results of our measurement of the Fo, F M, Fy/FM and Fy/Fo parameters support the above mentioned concept of resistance of the membrane processes to water stress. In the case of desiccating moss leaves, a more pronounced decrease in the Fy/FM ratio started at about 60 % RWC and in the case of barley at a very low level of about 30 % RWC (Fig. 3 C). Based on the inter-
404
MARTINA MATOUSKOV.A., HANA BARTOSKOVA, JAN NAus,
and
RADKO NOVOTNY
R punctatum
H vulgare A
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Time [s]
200 300
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Time [s]
pretation of this parameter as the maximal quantum efficiency of PSII photochemistry (Krause and Weis, 1991) it can be deduced that under our conditions of desiccation the function of PSII was inhibited in the later phases of water stress. In spite of the fact that the Fv/Fo ratio is a better indicator of changes in the rates of photosynthetic quantum conversion than Fy/FM (Babani and Lichtenthaler, 1996), its changes in the early phases of desiccation (to RWC about 50 %) were not significant (Fig. 3 D). Since the Fy/Fo ratio can serve as a fairly close measure of the potential photosynthetic capacity (Babani and Lichtenthaler, 1996), it appears that this characteristic was not affected upon the initial RWC decrease in both desiccating moss leaves and barley leaf segments. When the spectral ChI fluorescence parameters at 77 K were followed during desiccation, the F685/F735 ratio was relatively sensitive in the case of rapid water loss in R. punctatum leaves (Table 1). However, the significant decrease in this ratio was probably caused by the changes in leaf optical properties (see Results). Hence this ratio can give little information about the functional changes. Nevertheless, the F6851 F735 ratio might be used for a rough estimation of the water content in the fast desiccating moss leaves.
200 300
Fig. 4: Time dependencies of 1 - qP and qN in desiccating H. vulgare segments (A, C) and in R. punctatum leaves (B, D) within the first 5 min after switching on actinic PAR of 70 limol m -2 S-1 (barley) or 20 limol m- 2 S-1 (moss). The percentage numbers mean values of RWC. Arrows el) indicate the onset of actinic PAR. The first values of 1 - qP and qN (time <0 s) were measured for dark-adapted leaf samples. The following values were determined using saturating pulses. The first pulse was applied 2 s after switching on actinic PAR and the following ones in 20 s intervals. Each point represents a median calculated from seven measurements. Asterisks indicate statistically significant differences between hydrated (96 % RWC for barley and 100 % RWC for moss) and desiccating leaf samples (* P<0.05; ** P
It may be concluded that the parameters Fo, FM , Fy/FM and Fy/Fo have low sensitivity for detecting the early stages of the desiccation process. This insensitivity could be warranted by the fact that these fluorescence parameters reflect the state of dark-adapted photosynthetic apparatus and that slight functional changes due to the acclimation to the given water status need not be expressed in this state. The mentioned parameters can reveal only changes involving a more pronounced damage to the photosynthetic apparatus due to severe water stress (Havaux, 1992; Genty et aI., 1987). In a similar way, the steady-state or saturated values of the 1 - qP and qN quenching parameters were found to be of low sensitivity to the early stages of water stress, particularly in the case of the barley leaf segments (Fig. 4). These results are in accordance with those reported for Digitalis lanata (Stuhlfauth et aI., 1988), Helianthus annuus and Phaseolus vulgaris (Scheuermann et al., 1991) exposed to mild water stress. The steady-state values of these parameters reflect the state of the photosynthetic apparatus in the light-adapted leaf samples when a balance between processes of formation and consumption of L1pH occurs. It seems that in the steady-state, the changes caused by lower water content can be functionally compensated by some mechanisms (e.g. by opening of a
Dark-desiccation and Chi Fluorescence new route for utilization of electrons with a final acceptor other than CO 2 , probably molecular oxygen - Havaux, 1992) and are not reflected in the above mentioned fluorescence parameters. In contrast to the above discussed steady-state Chi fluorescence parameters those parameters detected during transitions of the photosynthetic system from the dark- to lightadapted state may be expected to be much more sensitive to the water status. Analysing the induction curves of the 1 - qP and qN parameters (Fig. 4) an interval can be found in which these parameters were very sensitive to the initial loss of water. For barley, this interval involved points measured in 22 and 42 s of the induction (Fig. 4 A, C). It is apparent that a slowdown of both the decrease in 1 - qP to steady-state and the increase in qN in the first minute of induction occurred with the RWC decline. As the character of the induction curves was different for R. punctatum (Fig. 4 B, D), the interval of sensitive differences was much wider and involved 1 - qP and qN values measured in 2, 22, 42 and 62 s of actinic illumination. The changes in 1 - qP and qN in this interval of Chi fluorescence induction occurred already in the initial phases of desiccation (at 69 % RWC in barley and 80 % RWC in moss). The loss of water evidently induced less favourable conditions for electron and proton transport and related quenching mechanisms (Dau, 1994), and the processes leading to steady-state were slowed down. The similar retardation of these processes has been shown also in desiccating Digitalis lanata (Stuhlfauth et al., 1988). Possible causes of this slowdown could be an impairment of electron flow from the plastoquinone pool via PSI and an inhibition of electron transport due to a reduced demand for NADPH and ATP in relation to drought induced changes in carbon metabolism (Genty et al., 1987; Ogren, 1990). These results indicate that the processes of transition of the photosynthetic apparatus from the dark- to light-adapted state were the most sensitive to the initial water loss and can serve as an indicator of a mild water stress. As shown above, these processes were sensitive in desiccating barley leaf segments as well as in the moss leaves irrespective of a very different rate of tissue water loss. Changes in these processes probably represent acclimation of photosynthetic function to the water status rather than damage to the photosynthetic apparatus.
Comparison of H. vulgare and R. punctatum responses to desiccation The different kinetics of the water loss from the tissue (Fig. 1) can be ascribed to the different leaf structures (Fig. 2) and consequently to a different heterogeneity in the release of water in barley and moss leaves. Based on knowledge of these leaf structures and previously reported results of other authors (Proctor, 1982; Rayan and Matsuda, 1988) it can be expected that the photosynthesizing cells in the barley leaf segments have kept water more effectively than in the moss leaves. This concept is in accordance with our observation that the dehydration of barley tissue did not influence Chi fluorescence considerably until the loss of most of the water (to 30 % RWC) , whereas in moss the more pronounced
405
changes in Chi fluorescence parameters started already at
60% RWC. The different character of changes in the Chi fluorescence parameters (Figs. 3, 4) caused by water loss in the barley and moss leaves may be related to the mentioned different rate of desiccation. In our opinion, this strongly different rate of water loss from photosynthesizing cells led to different strategies of the leaf reaction. These strategies are expressed more distinctly in the qualitatively different behaviour of Fa and F M . A slower water loss in the barley leaves could allow a partial acclimation of the thylakoid apparatus and conservation of the system. The high rate of water loss in the moss leaves led mostly to an inactivation of PSI! centres. It is not clear at this moment whether the distinct strategies of the two plant species are caused only by the kinetics of the water loss or are connected with a genetic difference of the species. To answer this question further research is required. Acknowledgements
We wish to thank Dr. Petr Ilik for critical reading of the manuscript and Jifi Skotnica and Jifi Fiala for technical assistance. This work was financed by grant No. 31503004/97 from the Faculty of Science, Palacky University.
References AGATI, G., F. FUSI, P. MAZZINGHI, and M. LipUCCI DI PAOLA: A simple approach to the evaluation of the reabsorption of chlorophyll fluorescence spectra in intact leaves. J. Phorochem. PhotobioI. B: BioI. 17, 163-171 (1993). ANDERSON, J. M., W S. CHOW, and D. J. GOODCHILD: Thylakoid membrane organisation in sun/shade acclimation. Aust. J. Plant Physiol. 15, 11-26 (1988). BABANI, F. and H. K. LICHTE NT HALER: Light-induced and agedependent development of chloroplasts in etiolated barley leaves as visualized by determination of photosynthetic pigments, CO 2 assimilation rates and different kinds of chlorophyll fluorescence ratios. J. Plant Physiol. 148,555-566 (1996). BUSCHMANN, C. and H. K. LICHTENTHALER: Principles and characteristics of multi-colour fluorescence imaging of plants. J. Plant Physiol. 152,297-314 (1998). CHEN, Y. H. and B. D. Hsu: Effect of dehydration on the electron transport of Chlorella. An in vivo fluorescence study. Photosynth. Res. 46, 295-299 (1995). CORNIC, G., J. GHASHGHAIE, B. GENTY, and J.-M. BRIANTAIS: Leaf photosynthesis is resistant to a mild drought stress. Photosynthetica 27, 295-309 (1992). DAu, H.: Molecular mechanisms and quantitative models of variable photosystem II fluorescence. Photochem. Photobiol. 60, 1-23 (1994). DILKS, T. ]. K. and M. C. F. PROCTOR: Photosynthesis, respiration and water content in btyophytes. New Phytol. 82,97-114 (1979). EICKMEIER, W. G., C. CASPER, and B. OSMOND: Chlorophyll fluorescence in the resurrection plant Selaginella lepidophylla (Hook. & Grev.) Spring during high-light and desiccation stress, and evidence for zeaxanthin-associated photoprotection. Planta 189, 30-38 (1993). FEEKES, W.: De tarwe en haar milieu. Verso XVII Tech. Tarwe Comm. Groningen, 560 (1941). GENTY, B., ].-M. BRIANTAIS, and ]. B. VIEIRA DA SILVA: Effects of drought on primaty photosynthetic processes of cotton leaves. Plant Physiol. 83,360-364 (1987).
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GITELSON, A. A., C. BUSCHMANN, and H. K. LICHTENTHALER: Leaf chlorophyll fluorescence corrected for re-absorption by means of absorption and reflectance measurements. ]. Plant Physiol. 152, 283-296 (1998). HAVAUX, M.: Stress tolerance of photosystem II in vivo. Antagonistic effects of water, heat, and photoinhibition stress. Plant Physiol. 100,424-432 (1992). HSIAO, T. Plant responses to water stress. Annu. Rev. Plant Physiol. 24, 519-570 (1973). KAISER, W. M.: Effects of water deficit on photosynthetic capacity. Physiol. Plant. 71, 142-149 (1987). KRAUSE, G. H. and E. WEIS: Chlorophyll fluorescence and photosynthesis: The basics. Annu. Rev. Plant Physiol. Plant Mol. BioI. 42,313-349 (1991). LAzAR, D. and]. NAUS: Statistical properties of chlorophyll fluorescence induction parameters. Photosynthetica 35, 121-127 (1998). LAZAR, D., ]. NAUS, M. MATOUSKOVA, and M. FLASAROVA: Mathematical modeling of changes in chlorophyll fluorescence induction caused by herbicides. Pestic. Biochem. Physiol. 57, 200-210 (1997). LEE, D. W., R. A. BONE, S. L. TARSIS, and D. STORCH: Correlates of optical properties in tropical forest sun and extreme-shade plants. Amer. J. Bot. 77, 370-380 (1990). LICHTENTHALER, H. K.: Chlorophylls and carotenoids. Pigments of photosynthetic biomembranes. Methods in Enzymol. 148, 350382 (1987 a). - Chlorophyll fluorescence signatures of leaves during the autumnal chlorophyll breakdown. ]. Plant Physiol. 131, 101-110 (1987b). LICHTENTHALER, H. K. and U. RrNDERLE: The role of chlorophyll fluorescence in the detection of stress conditions in plants. CRC Crit. Rev. Anal. Chern. 19, S29 - S85 (1988). MARTIN, C. E. and D. A. WARNER: The effects of desiccation on concentrations and alb ratios of chlorophyll in Leucobryum glaucum and Thuidium delicatulum. New Phytol. 96, 545-550 (1984). MERZLYAK, M. N. and A. GITELSON: Why and what for are the leaves yellow in autumn? On the interpretation of optical spectra of senescing leaves (Acer platanoides L.). ]. Plant Physiol. 145, 315-320 (1995). NAUS, J., T. KLINKOVSKY, P. ILiK, and D. CIKANEK: Model studies of chlorophyll fluorescence reabsorption at chloroplast level under different exciting conditions. Photosynth. Res. 40,67-74 (1994). OGREN, E.: Evaluation of chlorophyll fluorescence as a probe for drought stress in willow leaves. Plant Physiol. 93, 1280-1285 (1990).
c.:
PECIAR, v., M. CERVENKA, and E HINDAK: Oddelenie Bryophyta (Mechorasty). In: PECIAR, v., M. CERVENKA, and E HINDAK (eds.): ZakIady systemu a evohicie rytrusnych rastlin, pp. 387431. Bratislava (1984). PROCTOR, M. C. E: Physiological ecology: Water relations, light and temperature responses, carbon balance. In: SMITH, ]. A. E (ed.): Bryophyte Ecology, pp. 333-391. Chapman and Hall, London, New York (1982). - Structure and ecological adaptation. In: DRYER, A. F. and ]. G. DUCKETT (eds.): The Experimental Biology of Bryophytes, pp. 9-37. Academic Press, London (1984). RAYAN, A. and K. MATSUDA: The relation of anatomy to water movement and cellular response in young barley leaves. Plant Physiol. 87, 853-858 (1988). SCHEUERMANN, R., K. BIEHLER, T. STUHLFAUTH, and H. P. FOCK: Simultaneous gas exchange and fluorescence measurements indicate differences in the response of sunflower, bean and maize to water stress. Photosynth. Res. 27, 189-197 (1991). SCHREIBER, U. and W BILGER: Rapid assessment of stress effects on plant leaves by chlorophyll fluorescence measurements. In: TENHUNEN, ]., F. CATARINO, O. L. LANGE, and W. OECHEL (eds.): Plant Response to Stress, pp. 27- 53. Springer, Berlin (1987). SCHREIBER, u., U. SCHLIWA, and W. BILGER: Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth. Res. 10, 51-62 (1986). SCHWAB, K. B., U. SCHREIBER, and U. HEBER: Response of photosynthesis and respiration of resurrection plants to desiccation and rehydration. Planta 177, 217-227 (1989). SEEL, WE., N. R. BAKER, and J. A. LEE: Analysis of the decrease in photosynthesis on desiccation of mosses from xeric and hydric environments. Physiol. Plant. 86, 451-458 (1992). STRASSER, R. J., A. SRIVASTAVA, and GOVINDJEE: Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. Photochern. Photobiol. 61, 32-42 (1995). STUHLFAUTH, T., D. E SULTEMEYER, S. WEINZ, and H. P. FOCK: Fluorescence quenching and gas exchange in a water stressed C3 plant, Digitalis lanata. Plant Physiol. 86, 246-250 (1988). TUBA, Z., Z. CSINTALAN, and M. C. PROCTOR: Photosynthetic response of moss Tortula ruralis, ssp. ruralis, and the lichens Cladonia convoluta and C. furcata to water deficit and short periods of desiccation, and their ecophysiological significance: a baseline study at present-day CO 2 concentration. New Phytol. 133, 353361 (1996). VAN KOOTEN, O. and]. F. H. SNELL: The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth. Res. 25, 147-150 (1990).
j. Plant Physiol. Vol. 155. pp. 399-406 (1999) http://www.urbanfischer.de/journals/j pp
PIani PbysjllllY © 1999 URBAN & FISCHER
Reaction of Photosynthetic Apparatus to Dark Desiccation Sensitively Detected by the Induction of Chlorophyll Fluorescence Quenching 1 1 MARTINA MATOUSKOVA , HANA BARTOSKOVA , JAN NAUSl,
and
RADKO NOVOTNy.2
1
Department of Experimental Physics, Palacky University, ti'. Svobody 26, 77146 Olomouc, Czech Republic
2
Center for Microscopic Methods, Faculty of Medicine, Palacky University, 1. P. Pavlova 35, 77520 Olomouc, Czech Republic
Received September 14, 1998 . Accepted February 2, 1999
Summary
Chlorophyll fluorescence parameters Fo, FM, Fy/FM' Fy/Fo, the emission and excitation spectra and induction of fluorescence quenching coefficients qP and qN were followed during the desiccation of leaf segments of spring barley (Hordeum vulgare L. cv. Akcent) and detached leaves of an endohydric moss (Rhizomnium punctatum Hedw.) exposed to the same dark-desiccation conditions (35 % air relative humidity, 24°C). A sensitive fluorescence parameter in the initial phases of tissue desiccation was looked for. A decrease in relative water content (RWC) was much faster in the moss leaves (to 30 % RWC in 10 min) than in barley leaves (to about 30% RWC in 17h). A rather insensitive parameter, Fy/F M, started to decrease at 60 % RWC for the moss and at 30 % RWC for the barley. A more pronounced decrease in comparison with Fy/FM was observed in the case of the Fy/Fo. The ratio of bands (F 685/F 735) in the emission spectra at two excitation wavelengths (436 and 475 nm) was significantly changed only in desiccating R. punctatum. However, the decline in the F 685/F 735 ratio could be ascribed mostly to the increased fluorescence reabsorption. The excitation spectra were practically insensitive to the desiccation process in both species. Much more sensitive parameters reflecting the initial decrease in water content in the tissue were the qP and especially qN coefficients detected during induction elicited by the actinic light. When using the actinic irradiance for the induction similar to that of plant cultivation, the optimal interval for qp and qN detection in barley included values measured in 22 and 42 s of induction. The induction time interval of qP and qN sensitivity was much larger for the moss (included values measured in 2, 22, 42 and 62 s). Based on a distinct character of the induction curves and different changes in the Fo and FM parameters, different strategies of the barley and moss leaves in response to the same desiccation conditions were suggested.
Key words: Barley, chlorophyll fluorescence, desiccation, moss, relative water content. Abbreviations: Chi a and b = chlorophyll a and b; a + b and alb = sum and ratio of Chi a and b, respectively; Chi = chlorophyll; Fo = minimal Chi fluorescence intensity of the dark-adapted leaf; FM and FM' = maximal Chi fluorescence intensity of the dark- and light-adapted leaf; Fy/FM = (FM-FO)/FM = maximal quantum efficiency of photochemistry of PSII; LHC = light harvesting complex; PAR = photosynthetically active radiation; PSII = Photosystem II; OA = primary quinone acceptor ofPSII; qp and qN = photochemical and non-photochemical Chi fluorescence quenching coefficients; RWC = relative water content; x + c = total carotenoid content. 0176-1617/99/155/399 $ 12.00/0
400
MARTINA MATOUSKOVA, HANA BARTOSKOVA, JAN NAus, and RADKO NOVOTNY
Introduction
Responses of plants to the shortage of water leading to water stress are very variable and a variety of alterations in photosynthesis can be found (Hsiao, 1973). The extent of the effects of water stress is dependent on the intensity and duration of the stress as well as on the genetically determined capacity of plant species to cope with water deficit. It is thought (Schreiber and Bilger, 1987; Schwab et a!., 1989; Comic et a!., 1992) that in vascular plants the decrease in the rate of photosynthesis in initial phases of water stress is caused mainly by an inhibition of dark-reactions of photosynthesis. This inhibition occurs because of i) a decreasing intercellular CO 2 concentration due to stomatal closure (Kaiser, 1987) and ii) an inhibition of the activity of Calvin cycle enzymes (Schreiber and Bilger, 1987). A direct effect of desiccation on electron transport in photosystem I! (PSII) was observed when severe dehydration had been reached (see Comic et a!., 1992). In bryophytes the stomatal limitation of photosynthesis is impossible because bryophyte gametophytes have no stomata (Peciar et al., 1984). However, the other photosynthetic responses to water stress are similar to those of vascular plants (Tuba et a!., 1996). Measurement of the chlorophyll fluorescence induction curve is a very useful method for detection of changes in the photosynthetic apparatus due to stress constraints (e. g. Lichtenthaler and Rinderle, 1988). One of the parameters most widely studied with plants in general is Fy/F M , the maximal quantum efficiency of PSI! (Krause and Weis, 1991). This parameter has been found to be constant in initial phases of water stress in various types of plants (Havaux, 1992; Seel et a!., 1992; Eickmeier et a!., 1993; Chen and Hsu, 1995). In accordance with the above mentioned desiccation response it can be expected that the ChI fluorescence parameters reflecting electron transport beyond PSI! will be changed during mild dehydration rather than Fy/FM (Genty et a!., 1987; Stuhlfauth et a!., 1988). In this work, we used several ChI fluorescence methods for the study of the photosynthetic res po me of plants exposed to the same dark-desiccation conditions. We looked for a sensitive ChI fluorescence parameter changing in the initial phases of tissue dehydration and intended to compare its sensitivity in plants with different kinetics of water loss. For this reason we have chosen a monocotyledon Hordeum vulgare L. (cv. Akcent) and an endohydric moss Rhizomnium punctatum Hedw., two plants with different kinetics of desiccation. A sensitive time interval of an induction of quenching coefficients was found for the different physiological reactions of these plants.
Materials and Methods Plant material and stress treatment
Plams uf spring barley (Hordeum vulgare L. (v. Akccnt) were grown in a growth chamber at (20 ± 2) "C and relative air humidity (RH) of (60 ± 5) % on artificial soil composed of perlite and Knopp solution. The light regime was 8 h dark/16 h light (fluorescem light, Tesla Z 25W) with PAR intensity of 90 ~mol m 2 s-l The primary
leaves of plants in the growth phase 1.2 (two leaves) according to Feekes (1941) were used for the experiment. Gametophytes and sporophytes of the moss Rhizomnium punctatum (Hedw.) were collected in November 1997 near a small waterfall ReSov near Rymafov, north-east of the Czech Republic (49" 50' N, IT 15' E). Moss tutves together with a 0.5 em layer of soil were placed in covered Petri dishes in a growth chamber and kept under the above mentioned dark/light regime for 10 days. The RH of the air above the plants was 70 % and the cultivation PAR intensity was 10-15 ~mol m- 2 S-I. For sample preparation a 2-cm long segment of a barley leaf blade was cut off 1.5 cm from the tip of the leaf or several whole moss leaves were detached from the gametophytes. The samples were placed into Petri dishes and dried for 40 h (barley) or 1.5 h (moss) in stationary air in the dark. The conditions of the process of desiccation were T = (24 ± 1) "C and RH = (35 ± 1) % (Humidity and Temperature Meter HM 34C, Vaisala, Helsinki, Finland).
Determination of relative water content
The RWC of both barley and moss samples expressed in percentage of water-saturated tissue were calculated using the equation: RWC = ( mfresh-mdry ). 100 ffisaturated - ffidey
The msaturated of the barley segments was determined from the weight minit measured immediately after detachment of the segments by the relation msarurated = f . minit. The filling coefficient f was found by an independent measurement to be 1.03 ± 0.01. The fresh weight (mfresh) was estimated at a given time of desiccation and the dry mass weight (mdr ) after drying at 105 "C for 60 min. In the case of moss leaves the saturated weight (msarurated) was taken to be equal to the minit and the dry mass weight was determined after drying ofleaves at 105"C for 15 min. Light microscopy
Parts of the hydrated H. vulgare and R. punctatum leaves were fixed for 4 h in 3 % glutaraldehyde under vacuum and postfixed for 4 h in 1 % OS04 in 0.1 mol L -I phosphate buffer, pH 7.2. The samples were dehydrated in a graded acetone series with uranylacetate and embedded in Durcupan ACM (Fluka). The 1 ~m sections were mounted on slides and were stained with toluidine blue. The micrographs were made with a light microscope Jenamed Variant (Carl Zeiss Jena, Germany). Chl fluorescence measurements In vivo ChI a fluorescence was measured at room temperature with a modulation fluorometer PAM 2000 (Walz, Effeltrich, Germany). The leaf samples were dark-adapted (for a minimum time of 2.5 h) before the beginning of experiments. The initial Chi fluorescence intensity of the dark-adapted samples (Fo) was obtained upon excitation with a weak measuring beam (0.08 for H vulgare and 0.3 ~mol m- 2 S-I for R. punctatum). The higher intensity in the latter case was chosen to ensure a reliable signal. An 800 ms saturating pulse was used for determination of the maximal fluorescence intensity (F M ) of the dark-adapted samples. The correct intensity and length of the pulse was checked via kinetics of the Chi fluorescence response to the light pulse. Maximum ChI fluorescence intensity during the actinic illumination (F'M) was determined using additional saturating pulses (see Van Kooten and Snell, 1990). The first pulse was applied 2 s after switching on actinic PAR and the following ones in 20 s intervals. The 1 - qP parameter was calculated using the equation 1 - qp = 1 - (F' M- F,l/(F'M-F o), where Ft was the ChI
401
Dark-desiccation and Chi Fluorescence fluorescence intensity at the time t of the actinic illumination. The coefficient of non-photochemical quenching (qN) was calculated from the equation qN = (FM-F'M)/(FM-FO) (Schreiber et al., 1986). A general statistical description (medians and quartiles) was used in the case of the parameters of ChI fluorescence (Lazar and Naus, 1998). The segments of H. vulgare leaves placed in the Walz clips were irradiated by actinic PAR with an intensity of about 70 !lmol m -2 S-I. A leaf of R. punctatum was placed on a plastic pad and covered with a black mask with a round aperture (radius of 2 mm). The moss leaves were irradiated by actinic light of about 20 !lmol m- 1 S-I. The intensities of the actinic PAR were chosen to reach a comparable intensity to that of cultivation. The fast ChI fluorescence induction (the O-J-l-P transient) of the dark-adapted leaf samples was measured using a portable fluorometer PEA (Hansatech, Norfolk, England). The excitation intensity of PAR was 4,300 !lmol m -2 s-I and the exposure time was 2s. ChI fluorescence emission and excitation spectra at 77 K were measured using a Fluorescence Spectrophotometer F-4500 (Hitachi, Tokyo, Japan). The leaf samples were mounted on a holder and immersed into an optical Dewar flask with liquid nitrogen. The emission spectra (in a range from 650 to 800 nm) were measured at the excitation wavelengths of 436 nm (preferentially ChI a excited) and 475 nm (mainly ChI b together with carotenoids excited). The spectral halfWidths of the excitation and emission monochromators were 5 and 2.5 nm, respectively. The corrected excitation spectra (in a range from 400 to 550 nm) were measured at the emission wavelengths of 685 and 735 nm. The spectral halfWidths of the excitation and emission monochromators were 2.5 and 5 nm, respectively.
Pigment determination The contents of ChI a and b and the sum of carotenoids of the leaves were determined in 80 % acetone according to Lichtenthaler (1987a) using a DU-8 UV-VlSIBLE Spectrophotometer (Beckman, USA).
100
A 0
I
\
\
~( 10
20
20
40
o
20
40
60
80
Time [min]
Fig. 1: Changes in RWC with time for H. vulgare leaf segments (A) and detached leaves of R. punctatum (B) desiccated in the dark at 35 % RH and 24 ·C for 40 h (barley) and 90 min (moss), respectively. Means and SD are presented, n = 7 (barley) or n = 5 (moss).
Table 1: The effect of dark-desiccation on photosynthetic pigment content, pigment ratios, and ratios of bands in the emission and excitation ChI fluorescence spectra (77 K) of R. punctatum and H. vulgare leaves. The time needed to reach 8 % (moss) and 10 % RWC (barley) was 1.5 hand 40 h, respectively. The pigment values are given as mgg- I initial fresh weight. R. pUJ'/ctatum
Parameter
H. vulgare
RWC=IOO%
RWC=8%
RWC=96%
RWC= 10%
2.36±0.51 1.12±0.25 0.66±0.14 2.12±0.06 5.23±O.18
2.18±0.43 1.01±0.17 0.64±0.12 2.16±0.10 4.97±0.23
l.28±0.16 0.40±0.0,) 0.31 ±0.04 3.19±0.06 5.4HO.13
0.87±0.11'" 0.30±0.OS'" 0.29±0.03 2.94±0.22' 4.07±0.75"
1.04±0.21 l.3S±O.34 0.88±O.OS 1.1O±O.03
O.56±O.II" 0.67±O.16" 0.86±O.OS l.O2±O.03'"
0.34±0.01 0.39±O.OI 1.02±0.O3 0.90±0.O2
O.4HO.IS O.41±0.09 l.lHO.15 1.13±0.04'"
Pigment content
Chla (a) Chlb(b) alb
The decrease in relative water content of the barley leaf segments and detached moss leaves desiccating under the same external conditions was characterized by different kinetics (Fig. 1). The RWC decline in the barley segments was gradual within 40 h up to 10 % (Fig. 1 A). The initial water loss (to about 30 % RWC in 17 h) could correspond mostly to the loss of transpiration water and the water from the epidermal cells (Fig. 2 a). In the leaves of R. punctatum the RWC declined steeply during the first 20 min (to 30 % RWC in 10 min) to a value of 13 % (Fig. 1 B) and might be ascribed to the loss of symplastic water from the cells. The initial water loss was about 100 times faster in the moss leaves than in barley segments. A relatively slight following decline (to 8 % of RWC) was recorded during the next 70 min of dehydration and could be ascribed to the loss of apoplastic water from the cell walls (Dilks and Proctor, 1979; Proctor, 1982). Since leaves of R. punctatum are composed mostly of one or two cell layers and possess no epidermis and only a thin cuticle (Fig. 2 b) (Proctor, 1982; Peciar et aI., 1984; Proctor, 1984) the evaporation from all cells was very rapid.
30
40
I
Time[h]
(a+ b)/(x+c)
Kinetics ofthe RWC decrease
l . . . .,
,
o
60
l
\'
20
R. punctatum
i,
•
Carotcnoids x + c
Results
,
H. vulgare
\
40
80
\
~
u
s0::
• • •
\
;R 60 e.....
100
~
\
80
B
~
,
Chi fluorescence ratios F68S/F73S (exc. F685/1'735 (exc. E436/E475 (em. E436/E475 (em.
436 475 68') 73')
nm) nm) nm) nm)
Means and SD are shown, n :::: 7. Asterisks indicate statistically significant differences between hydrated and desiccated Ieafsamples (' P
Pigment content and pigment ratio Significant changes in pigment content and pigment ratios due to dehydration were observed only in the case of the barley leaf segments (Table 1). The desiccation of these segments was accompanied by a decrease in the chlorophyll content by about 30 %, whereas the total carotenoid content was maintained. Thus, the (a + b)/(x + c) ratio decreased during desiccation of the barley segments, which represents a similar tendency of the pigment breakdown as during autumnal senescence (Merzlyak and Gitelson, 1995). The slight decrease in the ChI alb ratio indicates that the content of ChI a declined more rapidly than the ChI b content. It should be noted here that the lower Chi alb ratio in hydrated moss leaves in comparison with barley indicates a shade adaptation of the moss plants (Anderson et aI., 1988; Martin and Warner, 1984).
402
MARTINA MATOUSKOVA, HANA BARTOSKOVA, JAN NAus,
and RADKO
b
Fig. 2: The light microscope phorographs of 111m leaf cross-sections of hydrated Hordeum vulgare (a) and Rhizomnium punctatum (b). E, epidermis; M, mesophyll; VB, vascular bundle. Bars 100 11m.
Chi fluorescence parameters Changes in the Fo, FM, Fv/FM and Fv/Fo parameters during desiccation of the moss leaves and barley leaf segments are shown in Fig. 3. The basic Fo and FM quantities are less precise as they reflect individual properties of the samples and may be affected also by the changing leaf structure due to desiccation. However, the changing leaf optical properties should change both quantities in a similar manner. This was not the case (Fig. 3A, B). In the desiccating moss leaves, Fo increased steeply about 2 times while FM remained constant (Fig. 3 A, B). The increase in Fo in the case of R. punctatum deserves more detailed attention. A slightly higher analytical PAR intensity than that of barley was used to ensure a reliable signal. It may be argued that this intensity of PAR might
NOVOTNY
have increased the measured Fo level due to OA reduction in some PSI! centres. Therefore, the Fo dependence was checked by a quite different method of Fo estimation from the very fast fluorescence induction kinetics (the O-J-I-P curve) measured by PEA, Hansatech (Strasser et aI., 1995; Lazar et aI., 1997). The increase in Fo was confirmed (Fig. 3 A dashed line). In contrast to moss, the desiccation of barley leaf segments led to only slight changes in Fo and to a decrease in FM to about 60 % of the initial level (Fig. 3A, B). It has been verified experimentally that the FM decrease in barley was not caused by a mere senescence in darkness (data not shown). Figure 3 C shows that the ratio Fv/FM declined during the desiccation of both H vulgare and R. punctatum. However, the rate and extent of this decline were different. In the case of moss the Fv/FM decline started at 60 % of RWC from the value of 0.71 and decreased to the value of 0.27 for 8 % RWC. The Fv/FM ratio of barley remained constant up to 30 % of RWC and then decreased from a value of 0.79 to a value of 0.59 for 10 % RWC. The value of Fv/FM of the most desiccated samples represented about 40 % (moss) and about 75 % (barley) of the Fv/F M value of the hydrated samples. In the case of the Fv/Fo ratio, a more pronounced decrease in comparison with Fv/FM was observed (Fig. 3 D). The values of Fv/Fo decreased in the most dehydrated samples to 25 % (moss) and 40 % (barley) of the values of the hydrated leaves. The ratio of fluorescence emission bands F685/F735 at two excitation wavelengths (436 and 475 nm) and the ratio of fluorescence excitation bands E436/E475 at two emission bands (F685 and F735) measured at 77K were also followed (Table 1). The most significant change found was the decrease in F685/F735 emission ratio in the case of R. punctatum upon desiccation to 8 % RWC by about 50 %. (The values of the band ratio were measured in 5 points of the dependence and they gradually decreased; only the first and last values are shown in Table 1.) This large change in the F685/F735 ratio without changes in the chlorophyll content could be explained by a strongly enhanced effect of Chi fluorescence reabsorption due to the clustering of chloroplasts in the dehydrated moss cytoplasm (Naus et aI., 1994). On the other hand, H vulgare revealed a tendency to increase this ratio. However, the desiccation of the barley segments was accompanied by the decrease in chlorophyll content by about 30 %. Thus, the reabsorption effect decreased due to a decrease in the chlorophyll concentration (Lichtenthaler, 1987b; Lichtenthaler and Rinderle, 1988; Buschmann and Lichtenthaler, 1998; Gitelson et aI., 1998). Although the effect of Chi fluorescence reabsorption dominates in the emission spectra the excitation spectra are not influenced because the absorbances at 436 and 475 nm are known to be similar (Lee et aI., 1990; Agati et aI., 1993). The changes in the Chi fluorescence excitation spectra were much lower and complicated to be interpreted as a complex combination of the optical, reabsorption and energy transfer changes that might have appeared. However, it seems that changes in energy transfer were not greater than 15 % based on the excitation spectra measurements. Thus, the excitation spectra in Table 1 help to exclude such mechanisms of the Fo increase and F685/F735 decrease in the R. punctatum leaves which are connected with significant changes in the energy transfer.
403
Dark-desiccation and Chi Fluorescence
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Fig. 3: Changes in the Chi fluorescence parameters Fo (A), FM (B), Fy/FM (C) and Fy/Fo (D) as measured with the PAM fluorometer by the application of a 0.8 s saturation pulse on darkadapted H. vulgare leaf segments and R. punctatum leaves during desiccation in the dark at 35 % RH and 24 'c. The values of Fo measured with the PEA instrument (Hansatech) in desiccating R. punctatum leaves are shown (- - - -... - - - -). Medians and quartiles are presented, n = 7. Asterisks indicate statistically significant differences between hydrated (96 % RWC for barley and 100 % RWC for moss) and desiccating leaf samples (* P<0.05; ** P
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The courses of 1 - qP and qN parameters during the first minutes of induction caused by actinic PAR are shown in Fig. 4. To measure the induction of quenching parameters qP and qN, we used the intensity of PAR close to the PAR of cultivation. This was done to avoid photo inhibition under too high intensity and insufficient excitation under too low PAR intensity. During the period of the actinic illumination the hydrated (non-stressed) H. vulgare as well as R. punctatum leaves exhibited characteristic induction kinetics of the 1 - qP and qN parameters (see Schreiber et aI., 1986). As is shown in Fig. 4, the changes in the induction curves due to desiccation were quite different for the barley and moss samples. In the case of barley, the steady-state values of 1- qp and qN differed only slightly upon the decrease in RWC from 96 % to 40 %. When RWC was 13 %, no decline in 1 - qP and only a gradual increase in qN without any relaxation were observed. In the moss leaves of 60 % RWC a decrease in 1 - qP within the first 2 min of illumination was followed by a gradual increase (Fig. 4 B). This effect was probably caused by a continual desiccation of the samples during the fluorescence induction (compare with Fig. 1 B). A similar effect was found in the case of qN in the moss leaves of 80 % and 60 % RWC (Fig. 4 D). When the RWC dropped to 40 %, only a progressive increase in both parameters was observed. The samples with 13 % RWC revealed no functional processes. The induction curves of the Chi fluorescence quenching in moss and barley leaves differed also in another aspect. In the
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case of H. vulgare only a tendency of changes in one direction was detectable. The qN coefficient increased in stages of induction near to steady state (3 to 5 min of induction). The qN parameter of R. punctatum increased already at 80 % RWC, reached its maximum for about 60 % RWC and decreased upon further drying. In the case of the moss leaves two phases of the desiccation effect were discernible.
Discussion
Sensitivity of Chi fluorescence parameters It has been reported that the photosynthetic processes related to thylakoid membranes are very resistant to drought conditions (Havaux, 1992; Schwab et aI., 1989) and that a mild water stress causes mainly an inhibition of dark-reactions of photosynthesis (Kaiser, 1987; Schreiber and Bilger, 1987). These conclusions result among others from measurements of chlorophyll fluorescence parameters whose changes started only when water stress became severe. Results of our measurement of the Fo, F M, Fy/FM and Fy/Fo parameters support the above mentioned concept of resistance of the membrane processes to water stress. In the case of desiccating moss leaves, a more pronounced decrease in the Fy/FM ratio started at about 60 % RWC and in the case of barley at a very low level of about 30 % RWC (Fig. 3 C). Based on the inter-
404
MARTINA MATOUSKOV.A., HANA BARTOSKOVA, JAN NAus,
and
RADKO NOVOTNY
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200 300
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pretation of this parameter as the maximal quantum efficiency of PSII photochemistry (Krause and Weis, 1991) it can be deduced that under our conditions of desiccation the function of PSII was inhibited in the later phases of water stress. In spite of the fact that the Fv/Fo ratio is a better indicator of changes in the rates of photosynthetic quantum conversion than Fy/FM (Babani and Lichtenthaler, 1996), its changes in the early phases of desiccation (to RWC about 50 %) were not significant (Fig. 3 D). Since the Fy/Fo ratio can serve as a fairly close measure of the potential photosynthetic capacity (Babani and Lichtenthaler, 1996), it appears that this characteristic was not affected upon the initial RWC decrease in both desiccating moss leaves and barley leaf segments. When the spectral ChI fluorescence parameters at 77 K were followed during desiccation, the F685/F735 ratio was relatively sensitive in the case of rapid water loss in R. punctatum leaves (Table 1). However, the significant decrease in this ratio was probably caused by the changes in leaf optical properties (see Results). Hence this ratio can give little information about the functional changes. Nevertheless, the F6851 F735 ratio might be used for a rough estimation of the water content in the fast desiccating moss leaves.
200 300
Fig. 4: Time dependencies of 1 - qP and qN in desiccating H. vulgare segments (A, C) and in R. punctatum leaves (B, D) within the first 5 min after switching on actinic PAR of 70 limol m -2 S-1 (barley) or 20 limol m- 2 S-1 (moss). The percentage numbers mean values of RWC. Arrows el) indicate the onset of actinic PAR. The first values of 1 - qP and qN (time <0 s) were measured for dark-adapted leaf samples. The following values were determined using saturating pulses. The first pulse was applied 2 s after switching on actinic PAR and the following ones in 20 s intervals. Each point represents a median calculated from seven measurements. Asterisks indicate statistically significant differences between hydrated (96 % RWC for barley and 100 % RWC for moss) and desiccating leaf samples (* P<0.05; ** P
It may be concluded that the parameters Fo, FM , Fy/FM and Fy/Fo have low sensitivity for detecting the early stages of the desiccation process. This insensitivity could be warranted by the fact that these fluorescence parameters reflect the state of dark-adapted photosynthetic apparatus and that slight functional changes due to the acclimation to the given water status need not be expressed in this state. The mentioned parameters can reveal only changes involving a more pronounced damage to the photosynthetic apparatus due to severe water stress (Havaux, 1992; Genty et aI., 1987). In a similar way, the steady-state or saturated values of the 1 - qP and qN quenching parameters were found to be of low sensitivity to the early stages of water stress, particularly in the case of the barley leaf segments (Fig. 4). These results are in accordance with those reported for Digitalis lanata (Stuhlfauth et aI., 1988), Helianthus annuus and Phaseolus vulgaris (Scheuermann et al., 1991) exposed to mild water stress. The steady-state values of these parameters reflect the state of the photosynthetic apparatus in the light-adapted leaf samples when a balance between processes of formation and consumption of L1pH occurs. It seems that in the steady-state, the changes caused by lower water content can be functionally compensated by some mechanisms (e.g. by opening of a
Dark-desiccation and Chi Fluorescence new route for utilization of electrons with a final acceptor other than CO 2 , probably molecular oxygen - Havaux, 1992) and are not reflected in the above mentioned fluorescence parameters. In contrast to the above discussed steady-state Chi fluorescence parameters those parameters detected during transitions of the photosynthetic system from the dark- to lightadapted state may be expected to be much more sensitive to the water status. Analysing the induction curves of the 1 - qP and qN parameters (Fig. 4) an interval can be found in which these parameters were very sensitive to the initial loss of water. For barley, this interval involved points measured in 22 and 42 s of the induction (Fig. 4 A, C). It is apparent that a slowdown of both the decrease in 1 - qP to steady-state and the increase in qN in the first minute of induction occurred with the RWC decline. As the character of the induction curves was different for R. punctatum (Fig. 4 B, D), the interval of sensitive differences was much wider and involved 1 - qP and qN values measured in 2, 22, 42 and 62 s of actinic illumination. The changes in 1 - qP and qN in this interval of Chi fluorescence induction occurred already in the initial phases of desiccation (at 69 % RWC in barley and 80 % RWC in moss). The loss of water evidently induced less favourable conditions for electron and proton transport and related quenching mechanisms (Dau, 1994), and the processes leading to steady-state were slowed down. The similar retardation of these processes has been shown also in desiccating Digitalis lanata (Stuhlfauth et al., 1988). Possible causes of this slowdown could be an impairment of electron flow from the plastoquinone pool via PSI and an inhibition of electron transport due to a reduced demand for NADPH and ATP in relation to drought induced changes in carbon metabolism (Genty et al., 1987; Ogren, 1990). These results indicate that the processes of transition of the photosynthetic apparatus from the dark- to light-adapted state were the most sensitive to the initial water loss and can serve as an indicator of a mild water stress. As shown above, these processes were sensitive in desiccating barley leaf segments as well as in the moss leaves irrespective of a very different rate of tissue water loss. Changes in these processes probably represent acclimation of photosynthetic function to the water status rather than damage to the photosynthetic apparatus.
Comparison of H. vulgare and R. punctatum responses to desiccation The different kinetics of the water loss from the tissue (Fig. 1) can be ascribed to the different leaf structures (Fig. 2) and consequently to a different heterogeneity in the release of water in barley and moss leaves. Based on knowledge of these leaf structures and previously reported results of other authors (Proctor, 1982; Rayan and Matsuda, 1988) it can be expected that the photosynthesizing cells in the barley leaf segments have kept water more effectively than in the moss leaves. This concept is in accordance with our observation that the dehydration of barley tissue did not influence Chi fluorescence considerably until the loss of most of the water (to 30 % RWC) , whereas in moss the more pronounced
405
changes in Chi fluorescence parameters started already at
60% RWC. The different character of changes in the Chi fluorescence parameters (Figs. 3, 4) caused by water loss in the barley and moss leaves may be related to the mentioned different rate of desiccation. In our opinion, this strongly different rate of water loss from photosynthesizing cells led to different strategies of the leaf reaction. These strategies are expressed more distinctly in the qualitatively different behaviour of Fa and F M . A slower water loss in the barley leaves could allow a partial acclimation of the thylakoid apparatus and conservation of the system. The high rate of water loss in the moss leaves led mostly to an inactivation of PSI! centres. It is not clear at this moment whether the distinct strategies of the two plant species are caused only by the kinetics of the water loss or are connected with a genetic difference of the species. To answer this question further research is required. Acknowledgements
We wish to thank Dr. Petr Ilik for critical reading of the manuscript and Jifi Skotnica and Jifi Fiala for technical assistance. This work was financed by grant No. 31503004/97 from the Faculty of Science, Palacky University.
References AGATI, G., F. FUSI, P. MAZZINGHI, and M. LipUCCI DI PAOLA: A simple approach to the evaluation of the reabsorption of chlorophyll fluorescence spectra in intact leaves. J. Phorochem. PhotobioI. B: BioI. 17, 163-171 (1993). ANDERSON, J. M., W S. CHOW, and D. J. GOODCHILD: Thylakoid membrane organisation in sun/shade acclimation. Aust. J. Plant Physiol. 15, 11-26 (1988). BABANI, F. and H. K. LICHTE NT HALER: Light-induced and agedependent development of chloroplasts in etiolated barley leaves as visualized by determination of photosynthetic pigments, CO 2 assimilation rates and different kinds of chlorophyll fluorescence ratios. J. Plant Physiol. 148,555-566 (1996). BUSCHMANN, C. and H. K. LICHTENTHALER: Principles and characteristics of multi-colour fluorescence imaging of plants. J. Plant Physiol. 152,297-314 (1998). CHEN, Y. H. and B. D. Hsu: Effect of dehydration on the electron transport of Chlorella. An in vivo fluorescence study. Photosynth. Res. 46, 295-299 (1995). CORNIC, G., J. GHASHGHAIE, B. GENTY, and J.-M. BRIANTAIS: Leaf photosynthesis is resistant to a mild drought stress. Photosynthetica 27, 295-309 (1992). DAu, H.: Molecular mechanisms and quantitative models of variable photosystem II fluorescence. Photochem. Photobiol. 60, 1-23 (1994). DILKS, T. ]. K. and M. C. F. PROCTOR: Photosynthesis, respiration and water content in btyophytes. New Phytol. 82,97-114 (1979). EICKMEIER, W. G., C. CASPER, and B. OSMOND: Chlorophyll fluorescence in the resurrection plant Selaginella lepidophylla (Hook. & Grev.) Spring during high-light and desiccation stress, and evidence for zeaxanthin-associated photoprotection. Planta 189, 30-38 (1993). FEEKES, W.: De tarwe en haar milieu. Verso XVII Tech. Tarwe Comm. Groningen, 560 (1941). GENTY, B., ].-M. BRIANTAIS, and ]. B. VIEIRA DA SILVA: Effects of drought on primaty photosynthetic processes of cotton leaves. Plant Physiol. 83,360-364 (1987).
406
MARTINA MATOUSKOVA, HANA BARTOSKOVA, ]AN NAUS, and RADKO NOVOTNY
GITELSON, A. A., C. BUSCHMANN, and H. K. LICHTENTHALER: Leaf chlorophyll fluorescence corrected for re-absorption by means of absorption and reflectance measurements. ]. Plant Physiol. 152, 283-296 (1998). HAVAUX, M.: Stress tolerance of photosystem II in vivo. Antagonistic effects of water, heat, and photoinhibition stress. Plant Physiol. 100,424-432 (1992). HSIAO, T. Plant responses to water stress. Annu. Rev. Plant Physiol. 24, 519-570 (1973). KAISER, W. M.: Effects of water deficit on photosynthetic capacity. Physiol. Plant. 71, 142-149 (1987). KRAUSE, G. H. and E. WEIS: Chlorophyll fluorescence and photosynthesis: The basics. Annu. Rev. Plant Physiol. Plant Mol. BioI. 42,313-349 (1991). LAzAR, D. and]. NAUS: Statistical properties of chlorophyll fluorescence induction parameters. Photosynthetica 35, 121-127 (1998). LAZAR, D., ]. NAUS, M. MATOUSKOVA, and M. FLASAROVA: Mathematical modeling of changes in chlorophyll fluorescence induction caused by herbicides. Pestic. Biochem. Physiol. 57, 200-210 (1997). LEE, D. W., R. A. BONE, S. L. TARSIS, and D. STORCH: Correlates of optical properties in tropical forest sun and extreme-shade plants. Amer. J. Bot. 77, 370-380 (1990). LICHTENTHALER, H. K.: Chlorophylls and carotenoids. Pigments of photosynthetic biomembranes. Methods in Enzymol. 148, 350382 (1987 a). - Chlorophyll fluorescence signatures of leaves during the autumnal chlorophyll breakdown. ]. Plant Physiol. 131, 101-110 (1987b). LICHTENTHALER, H. K. and U. RrNDERLE: The role of chlorophyll fluorescence in the detection of stress conditions in plants. CRC Crit. Rev. Anal. Chern. 19, S29 - S85 (1988). MARTIN, C. E. and D. A. WARNER: The effects of desiccation on concentrations and alb ratios of chlorophyll in Leucobryum glaucum and Thuidium delicatulum. New Phytol. 96, 545-550 (1984). MERZLYAK, M. N. and A. GITELSON: Why and what for are the leaves yellow in autumn? On the interpretation of optical spectra of senescing leaves (Acer platanoides L.). ]. Plant Physiol. 145, 315-320 (1995). NAUS, J., T. KLINKOVSKY, P. ILiK, and D. CIKANEK: Model studies of chlorophyll fluorescence reabsorption at chloroplast level under different exciting conditions. Photosynth. Res. 40,67-74 (1994). OGREN, E.: Evaluation of chlorophyll fluorescence as a probe for drought stress in willow leaves. Plant Physiol. 93, 1280-1285 (1990).
c.:
PECIAR, v., M. CERVENKA, and E HINDAK: Oddelenie Bryophyta (Mechorasty). In: PECIAR, v., M. CERVENKA, and E HINDAK (eds.): ZakIady systemu a evohicie rytrusnych rastlin, pp. 387431. Bratislava (1984). PROCTOR, M. C. E: Physiological ecology: Water relations, light and temperature responses, carbon balance. In: SMITH, ]. A. E (ed.): Bryophyte Ecology, pp. 333-391. Chapman and Hall, London, New York (1982). - Structure and ecological adaptation. In: DRYER, A. F. and ]. G. DUCKETT (eds.): The Experimental Biology of Bryophytes, pp. 9-37. Academic Press, London (1984). RAYAN, A. and K. MATSUDA: The relation of anatomy to water movement and cellular response in young barley leaves. Plant Physiol. 87, 853-858 (1988). SCHEUERMANN, R., K. BIEHLER, T. STUHLFAUTH, and H. P. FOCK: Simultaneous gas exchange and fluorescence measurements indicate differences in the response of sunflower, bean and maize to water stress. Photosynth. Res. 27, 189-197 (1991). SCHREIBER, U. and W BILGER: Rapid assessment of stress effects on plant leaves by chlorophyll fluorescence measurements. In: TENHUNEN, ]., F. CATARINO, O. L. LANGE, and W. OECHEL (eds.): Plant Response to Stress, pp. 27- 53. Springer, Berlin (1987). SCHREIBER, u., U. SCHLIWA, and W. BILGER: Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth. Res. 10, 51-62 (1986). SCHWAB, K. B., U. SCHREIBER, and U. HEBER: Response of photosynthesis and respiration of resurrection plants to desiccation and rehydration. Planta 177, 217-227 (1989). SEEL, WE., N. R. BAKER, and J. A. LEE: Analysis of the decrease in photosynthesis on desiccation of mosses from xeric and hydric environments. Physiol. Plant. 86, 451-458 (1992). STRASSER, R. J., A. SRIVASTAVA, and GOVINDJEE: Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. Photochern. Photobiol. 61, 32-42 (1995). STUHLFAUTH, T., D. E SULTEMEYER, S. WEINZ, and H. P. FOCK: Fluorescence quenching and gas exchange in a water stressed C3 plant, Digitalis lanata. Plant Physiol. 86, 246-250 (1988). TUBA, Z., Z. CSINTALAN, and M. C. PROCTOR: Photosynthetic response of moss Tortula ruralis, ssp. ruralis, and the lichens Cladonia convoluta and C. furcata to water deficit and short periods of desiccation, and their ecophysiological significance: a baseline study at present-day CO 2 concentration. New Phytol. 133, 353361 (1996). VAN KOOTEN, O. and]. F. H. SNELL: The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth. Res. 25, 147-150 (1990).