**3. Results**

The relative water content (RWC) was measured in the leaves from *Chrysanthemum morifolium* and *Spathiphyllum wallisii* plants at the start the experiment (control) and immediately after exposure to each stress photoperiod (Figure 1). The RWC values

*Chrysanthemum morifolium* (sun plant) and *Spathiphyllum wallisii* (shade plant) were grown in 500 mL pots at 22-25 ºC in the greenhouse with a natural photoperiod, under daytime irradiation maxima of around 800 and 200 mol·m-2·s-1 PPFD (sun and shade plants, respectively) and controlled watering to avoid drought stress (control conditions). To simulate stress conditions, adult plants were transferred to cultivation chambers and exposed to 18 h photoperiods of high light intensity (1060 mol·m-2·s-1 PPFD) supplied by a 100 W Flood Osram (Augsburg, Germany) white light lamp, at 35 ºC, followed by 6h nightperiods at 24 ºC, decreasing the irrigation to 50 mL/day, which was applied after the start

Plant water status was estimated by measuring the relative water content of leaves (RWC). The leaves were collected and immediately weighed to determine fresh weight (FW). They were then re-hydrated for 24 h at 4 ºC in darkness to determine the turgid weight (TW), and subsequently oven-dried for 24 h at 85 ºC to determine the dry weight (DW). The RWC was

Total Chlorophyll and carotenoids were determined by Lichtenthaler & Wellburn´s

Chlorophyll fluorescence was imaged, using the MINI-version of the Imaging-PAM (Heinz Walz GmbH, Effeltrich, Germany), in selected leaves attached to plants for the control and stress conditions and the measurements were made after the last night-period. The fluorometer used employs the same blue LEDs for the pulse modulated measuring light, continuous actinic illumination and saturation pulses. The minimal fluorescence yield (F0) and the maximal fluorescence yield (Fm), were measured in dark-adapted samples. F0 was measured at a low frequency of pulse modulated measuring light, while Fm was measured with the help of a saturation pulse. The maximal quantum yield of PS II was calculated as

Light response curves were realised illuminating the selected leaves with actinic light of different intensities (20, 41, 76, 134, 205, 249, 300, 371, 456, 581,726 mol·m-2·s-1 PPFD), with 2 min illumination periods at each intensity. After each illumination periods a saturation pulse was applied to determine the relative electron transport rate, the effective PS II quantum yield of illuminated samples ((Fm'-F)/Fm') and non-photochemical quenching (Nq)), all of which were automatically calculated by the ImagingWin software. Results are shown as color-coded images of the maximal quantum yield of PS II (Fv/Fm), the fluorescence yield (F), the effective PS II quantum yield of illuminated samples ((Fm'-F)/Fm')

The relative water content (RWC) was measured in the leaves from *Chrysanthemum morifolium* and *Spathiphyllum wallisii* plants at the start the experiment (control) and immediately after exposure to each stress photoperiod (Figure 1). The RWC values

**2. Materials and methods** 

the night period.

Fv/Fm = (Fm-F0)/Fm.

**3. Results** 

**2.1 Plant material and growth conditions** 

**2.2 Plant water status and pigments measurements** 

determined as 100 x (FW-DW)/ (TW-DW).

method using 80 % (v/v) acetone as solvent.

and non-photochemical quenching (Nq)).

**2.3 Chlorophyll fluorescence measurements** 

decreased in both species to around 60 % after the two stress photoperiods with low watering, indicating that the plants were subjected to moderate water deficit.

Fig. 1. The relative water content (RWC) of leaves from *Chrysanthemum morifolium* and *Spathiphyllum wallisii* plants in control conditions (C) and after exposure to one (1S) and two (2S) stress photoperiods (18 h, 1060 mol·m-2·s-1 PPFD, 35 ºC and low watering). The stress photoperiods were separated by 6 h night-periods at 24 ºC. The values are means SE from four independent experiments.

The fluorescence imaging technique was used to assess photosynthesis in sun and shade plants. Figure 2 shows the images of the maximal quantum yield of PS II (Fv/Fm) from a typical leaf, and the means values SE of *C. morifolium* and *S. wallisii* plants in control conditions and exposed to one and two stress photoperiods. The results are shown as colour-coded images according to the pattern shown below the images. All the leaves provided similar images with a homogeneous colour throughout the leaf. The mean Fv/Fm values in all cases were higher than 0.74, indicating that maximal quantum yield of PS II in leaves from *C. morifolium* and *S. wallisii* plants, in control conditions and exposed to one and two stress photoperiods, was quite normal (Krause & Weis, 1991; Schereiber et al., 1997) and that the maximal photosynthetic capacity of PS II in these species was probably unaffected by the stress condition used here; furthermore it seems that the photosynthetic apparatus is protected by mechanisms that dissipate excess excitation energy.

Figure 3 shows the amounts of total chlorophylls and carotenoids in leaves from *C. morifolium* and *S. wallisii* plants in control conditions and exposed to one and two stress photoperiods. No significant difference was observed between the control plants and those exposed to stress photoperiods in either sun or shade plants.

Using Chlorophyll Fluorescence Imaging for Early Assessment of

plants of both species exposed to stress photoperiods.

0

20

40

ETR

six independent experiments.

shown in the histograms.

Photosynthesis Tolerance to Drought, Heat and High Illumination 213

Figure 4 shows the light response curves for the relative electron transport rate in leaves from *C. morifolium* and *S. wallisii* plants in control conditions and exposed to one and two stress photoperiods. In both species, when light was not excessive, the relationship between the relative electron transport rate and the light intensity was linear (optimum line, Fig. 4). When the light became excessive, the relative electron transport rate decreased below the values predicted by the optimum line. Finally, when the photonic flux density was increased, a satured rate was reached, which represents the capacity of photosynthetic electron transport (Schreiber et al., 1997). In low light intensity of less than 100 mol·m-2·s-1, the relative electron transport rate was similar in control and stress-exposed *C. morifolium*  plants, but not in *S. wallisii*, where the values in plants exposed to stress photoperiods were lower than those predicted by the optimum line. the capacity of photosynthetic electron transport was greater in *C. morifolium* than in *S. wallisii* control plants and decreased in the

60 optimum line optimum line

*C. morifolium S. wallisii*

 C 1S 2S

0 100 300 500 700 0 100 300 500 700

Fig. 4. Light response curves for the relative electron transport rate (ETR) in intact darkadapted leaves of *Chrysanthemum morifolium* and *Spathiphyllum wallisii* plants in control conditions (green graphics) and exposed to one (blue graphics) and two (red graphics) stress

photoperiods were separated by 6 h night-periods at 24 ºC. The values are means SE from

Figure 5 shows the images obtained at two light intensities (20 and 300 mol·m-2·s-1) of the effective PS II quantum yield (Y(II)), the fluorescence yield (F) and non-photochemical quenching (Nq)) from a typical leaf of *C. morifolium* and *S. wallisii* plants in control conditions and exposed to one and two stress photoperiods. For comparison purposes, the data from the analysed entire leaves were also averaged and the medium values SE are

Leaves from the control plants and those exposed to stress photoperiods showed changes in the images of the fluorescence parameters in both sun and shade species illuminated with low and high light intensity. With low illumination (20 mol·m-2·s-1) the photochemical efficiency of control plants was approximately 0.5 and the leaves provided Y(II) images of a green-blue colour in both species; the fluorescence emission of control plants was higher in sun (0.32) than in shade species (0.21), while Nq was lower in sun plants (0.16, yellow

photoperiods (18 h, 1060 mol·m-2·s-1 PPFD, 35 ºC and low watering). The stress

PPFD (μmol · m-2 · s-1)

Fig. 2. Images and values of the maximal quantum yield of PS II (Fv/Fm) from typical leaves of *Chrysanthemum morifolium* and *Spathiphyllum wallisii* plants in control conditions (C) and exposed to one (1S) and two (2S) stress photoperiods (18 h, 1060 mol·m-2·s-1 PPFD, 35 ºC and low watering). The stress photoperiods were separated by 6 h night-periods at 24 ºC. Images are colour coded according to the pattern (0 to 1 x 100 range) shown below the images. The figure shows representative images from four independent experiments and the values are means SE from four different entire leaves.

Fig. 3. Total chlorophyll and carotenoids of leaves from *Chrysanthemum morifolium* and *Spathiphyllum wallisii* plants in control conditions (C) and exposed to one (1S) and two (2S) stress photoperiods (18 h, 1060 mol·m-2·s-1 PPFD, 35 ºC and low watering). The stress photoperiods were separated by 6 h night-periods at 24 ºC. The values are means SE from four independent experiments.

**C**

**1S**

**2S**

*C. morifolium S. wallisii*

0.778 ± 0.004 0.765± 0.002

0.751 ± 0.007 0.769±0.004

0.741 ± 0.009 0.762 ± 0.004

values are means SE from four different entire leaves.

Pigment concentration (mg·L-1)

0

four independent experiments.

5

10

15

20

25

**0 100**

Fig. 2. Images and values of the maximal quantum yield of PS II (Fv/Fm) from typical leaves of *Chrysanthemum morifolium* and *Spathiphyllum wallisii* plants in control conditions (C) and exposed to one (1S) and two (2S) stress photoperiods (18 h, 1060 mol·m-2·s-1 PPFD, 35 ºC and low watering). The stress photoperiods were separated by 6 h night-periods at 24 ºC. Images are colour coded according to the pattern (0 to 1 x 100 range) shown below the images. The figure shows representative images from four independent experiments and the

*C. morifolium S. wallisii*

Chlorophylls Carotenoids

Fig. 3. Total chlorophyll and carotenoids of leaves from *Chrysanthemum morifolium* and *Spathiphyllum wallisii* plants in control conditions (C) and exposed to one (1S) and two (2S) stress photoperiods (18 h, 1060 mol·m-2·s-1 PPFD, 35 ºC and low watering). The stress photoperiods were separated by 6 h night-periods at 24 ºC. The values are means SE from

C 1S 2S C 1S 2S

Figure 4 shows the light response curves for the relative electron transport rate in leaves from *C. morifolium* and *S. wallisii* plants in control conditions and exposed to one and two stress photoperiods. In both species, when light was not excessive, the relationship between the relative electron transport rate and the light intensity was linear (optimum line, Fig. 4). When the light became excessive, the relative electron transport rate decreased below the values predicted by the optimum line. Finally, when the photonic flux density was increased, a satured rate was reached, which represents the capacity of photosynthetic electron transport (Schreiber et al., 1997). In low light intensity of less than 100 mol·m-2·s-1, the relative electron transport rate was similar in control and stress-exposed *C. morifolium*  plants, but not in *S. wallisii*, where the values in plants exposed to stress photoperiods were lower than those predicted by the optimum line. the capacity of photosynthetic electron transport was greater in *C. morifolium* than in *S. wallisii* control plants and decreased in the plants of both species exposed to stress photoperiods.

Fig. 4. Light response curves for the relative electron transport rate (ETR) in intact darkadapted leaves of *Chrysanthemum morifolium* and *Spathiphyllum wallisii* plants in control conditions (green graphics) and exposed to one (blue graphics) and two (red graphics) stress photoperiods (18 h, 1060 mol·m-2·s-1 PPFD, 35 ºC and low watering). The stress photoperiods were separated by 6 h night-periods at 24 ºC. The values are means SE from six independent experiments.

Figure 5 shows the images obtained at two light intensities (20 and 300 mol·m-2·s-1) of the effective PS II quantum yield (Y(II)), the fluorescence yield (F) and non-photochemical quenching (Nq)) from a typical leaf of *C. morifolium* and *S. wallisii* plants in control conditions and exposed to one and two stress photoperiods. For comparison purposes, the data from the analysed entire leaves were also averaged and the medium values SE are shown in the histograms.

Leaves from the control plants and those exposed to stress photoperiods showed changes in the images of the fluorescence parameters in both sun and shade species illuminated with low and high light intensity. With low illumination (20 mol·m-2·s-1) the photochemical efficiency of control plants was approximately 0.5 and the leaves provided Y(II) images of a green-blue colour in both species; the fluorescence emission of control plants was higher in sun (0.32) than in shade species (0.21), while Nq was lower in sun plants (0.16, yellow

Using Chlorophyll Fluorescence Imaging for Early Assessment of

the values were higher in sun than in shade species.

**4. Discussion** 

2004; Teicher et al., 2000).

Photosynthesis Tolerance to Drought, Heat and High Illumination 215

(yellow-green images). When the same leaves were illuminated with 300 mol·m-2·s-1, the Y (II) decreased in both species, although values were higher in sun than in shade species, and the *C. morifolium* images showed orange-red colour in control leaves, which changed to red

The F emission decreased in sun plants and the images showed orange and orange-green colours, whereas in shade plants the F emission decreased in leaves of the control plants, but after stress photoperiods the leaf images showed only slight differences from those illuminated with 20 mol·m-2·s-1. The Nq increased significantly in both species, although

Fluorescence imaging represents a simple and non-invasive tool for the early detection of effects caused by adverse factors, which affect photosynthesis causing an imbalance in the processes of excitation energy dissipation (Long et al., 1994). This technique permits us to compare, by means of imagines, the variation in these processes and to study any damage caused in the same leaf as time progresses. Usually, changes in Fv/Fm of leaves adapted to dark, which represents the maximal quantum yield of PS II (Krause & Weis, 1991), are used as an indicator of the functional state of the photosynthetic apparatus (Barbagallo et al., 2003; Krause & Jahns, 2004; Oxborough, 2004b), since this parameter, which has a value of between 0.70 and 0.85 in unstressed leaves, falls under the influence of adverse factors (Ehlert & Hincha, 2008; Havaux & Lannoye, 1985; Joshi & Mohantly, 2004; Quiles & López,

Sun plants (*C. morifolium*) and shade plants (*S. wallisii*) were exposed to photoperiods with low watering, high illumination and heat. Even after two stress photoperiods no visible damage was observed in either plant species (not shown). Neither did the concentration of photosynthetic pigments or the Fv/Fm values show any significant decrease after the stress photoperiods, suggesting that chloroplasts are protected by mechanisms that dissipate excess excitation energy to prevent damage to the photosynthetic apparatus under adverse conditions. In this respect, we have reported that chlororespiration and cyclic electron flow pathways are involved in the tolerance to adverse factors in both sun and shade species (Díaz et al., 2007; Gamboa et al., 2009; Ibañez et al., 2010; Quiles, 2006; Tallón & Quiles, 2007). However, when the light response curves for the relative electron transport rate were depicted, differences were observed between control plants and those exposed to stress photoperiods, the capacity of photosynthetic electron transport being lower in plants exposed to stress photoperiods in both species. In *C. morifolium* after one or two stress photoperiods, the values were similar and the capacity of photosynthetic electron transport was approximately 22% lower than in control plants. However, in *S. wallisii* differences between plants exposed to one and two stress photoperiods were observed and the capacity of photosynthetic electron transport after one and two stress photoperiods was

The images of the fluorescence yield, the effective PS II quantum yield or photochemical efficiency and the non-photochemic quenching of fluorescence, which represents the heat dissipation in the antenna system (Müller et al., 2001), also showed significant differences, indicating that plants exposed to stress photoperiods behaved differently as regards the processes of dissipation of excitation energy, in each species. At low illumination (20 mol·m-2·s-1), fluorescence emission predominates over heat dissipation in the sun species, while the contrary occurs in the shade species, heat dissipation predominates over

approximately 27 and 44%, respectively, lower than that of control plants.

after two stress photoperiods, while *S. wallisii* images showed red colour in all cases.

Fig. 5. Images at 20 and 300 mol·m-2·s-1 PPFD of the effective PS II quantum yield (Y(II)), the fluorescence yield (F) and non-photochemical quenching (Nq) from a typical leaf attached to *Chrysanthemum morifolium* and *Spathiphyllum wallisii* plants, in control conditions (C) and exposed to one (1S) and two (2S) stress photoperiods (18 h, 1060 mol·m-2·s-1 PPFD, 35 ºC and low watering). The stress photoperiods were separated by 6 h night-periods at 24 ºC. Images are colour coded according to the pattern (0 to 1 x 100 range) shown below the images. The histograms show the means SE of parameters calculated from variable chlorophyll fluorescence measurements in six entire leaves.

images) than in shade plants (0.28, green images). After one and two stress photoperiods, the effective PSII quantum yield and the fluorescence emission decreased, moreso in shade than in sun plants, while Nq increased, moreso in shade (blue images) than in sun plants (yellow-green images). When the same leaves were illuminated with 300 mol·m-2·s-1, the Y (II) decreased in both species, although values were higher in sun than in shade species, and the *C. morifolium* images showed orange-red colour in control leaves, which changed to red after two stress photoperiods, while *S. wallisii* images showed red colour in all cases.

The F emission decreased in sun plants and the images showed orange and orange-green colours, whereas in shade plants the F emission decreased in leaves of the control plants, but after stress photoperiods the leaf images showed only slight differences from those illuminated with 20 mol·m-2·s-1. The Nq increased significantly in both species, although the values were higher in sun than in shade species.
