**6.2 Influence of the combined impact of stresses on soybean plants**

The third part of experiments aimed at assessing of the impact of the single stress factor salinity and combined stress factors salinity and enhanced UV-B radiation on young nitrogen fixing soybean plants. Spectral data were taken for 25 areas (pixels) of leaves from each group of investigated plants. Fig. 16 shows the averaged spectral reflectance characteristics of the control group and salinity treated at two NaCl concentrations nitrogen fixing soybean plants (Krezhova et al., 2011a). It is seen that changes in SRC for the salinized plants are noticeable in all investigated spectral ranges. The effect of the salinity was manifested by decreasing of the values in the green (520-580 nm) and red (640-680 nm) ranges and increasing in the NIR (720-770 nm). The red edge position of the SRC at low and high NaCl treatments is shifted to longer wavelengths (1 nm and 5 nm, respectively). On some of the leaves treated with 80 mM NaCl concentration the necroses spots were observed (Fig. 9a).

The results from the statistical analysis applying Student's t-criterion are shown in Table 11. Statistically significant differences are obtained at p<0.05 in the two cases of salinity treatment with the exception of 40 mM NaCl concentration in λ6 and λ8, and 80 mM NaCl in λ7.

Statistically significant differences between data means at wavelengths λi and λic, i = 1,…, 5 were established by the Student's t-criterion at p<0.05 for the data at the first three wavelengths and for both the NaCl concentrations. DA confirmed these findings in one

NaCl 40 mM NaCl 80 mM

Number of incorrectly classified objects

Table 10. Significance p-level of the linear DA for the set of amplitudes of the fluorescence

The results revealed that the two NaCl concentrations applied produce statistically significant changes in the forefront of leaf fluorescence spectra of the nitrogen fixing soybean plants. This corresponds to the salinity stress disclosed by the biochemical parameters (stress markers and pigments) and by the spectral reflectance in the VIS and NIR spectral ranges evaluated for the same soybean plants. The two remote sensing techniques (chlorophyll fluorescence and spectral reflectance) independently detected that both the

The third part of experiments aimed at assessing of the impact of the single stress factor salinity and combined stress factors salinity and enhanced UV-B radiation on young nitrogen fixing soybean plants. Spectral data were taken for 25 areas (pixels) of leaves from each group of investigated plants. Fig. 16 shows the averaged spectral reflectance characteristics of the control group and salinity treated at two NaCl concentrations nitrogen fixing soybean plants (Krezhova et al., 2011a). It is seen that changes in SRC for the salinized plants are noticeable in all investigated spectral ranges. The effect of the salinity was manifested by decreasing of the values in the green (520-580 nm) and red (640-680 nm) ranges and increasing in the NIR (720-770 nm). The red edge position of the SRC at low and high NaCl treatments is shifted to longer wavelengths (1 nm and 5 nm, respectively). On some of the leaves treated with 80 mM NaCl concentration the necroses spots were observed

The results from the statistical analysis applying Student's t-criterion are shown in Table 11. Statistically significant differences are obtained at p<0.05 in the two cases of salinity treatment with the exception of 40 mM NaCl concentration in λ6 and λ8, and 80 mM NaCl

NaCl concentrations bring to salinity stress in the nitrogen fixing soybean plants.

**6.2 Influence of the combined impact of stresses on soybean plants** 

λ<sup>1</sup> <0.001 8 from 40 <0.001 4 from 34 λ<sup>2</sup> <0.001 10 from 40 <0.001 5 from 34 λ<sup>3</sup> 0.0016 11 from 40 <0.001 8 from 34 λ<sup>4</sup> 0.318 15 from 40 0.2305 14 from 34 λ<sup>5</sup> 0.480 18 from 40 0.4919 12 from 34

NaCl 80 mM NaCl

Number of incorrectly classified objects

*pDA*

dimensional spaces defined separately by each of the five wavelengths.

40 mM

*pDA*

One dimensional spaces

spectra.

(Fig. 9a).

in λ7.

Fig. 16. Averaged spectral reflectance characteristics of control and treated with 40 mM and 80 mM NaCl nitrogen fixing soybean plants.


Table 11. Significance p-level of the t-criterion in the cases of 40 mm NaCl and 80 mm NaCl salinity.

Fig. 17 shows the averaged SRC of leaves of plants from the second set of three groups including the control (treated only with UV-B radiation) and the other two groups on which the combined action of stresses, salinity at two concentrations + UV-B radiation, was applied. The values of the averaged spectral characteristics of treated leaves with respect to control decrease significantly in the green and red (520-660 nm), and NIR ranges. For these SRC it is observed an approaching of the red edge position nearer to the control (2 nm), which is an indicator for diminishing effect of the salinity stress. Averaged SRC after (80 mM NaCl + UV-B) treatment is very close to the one after (40 mM NaCl + UV-B) treatment.

Spectral Remote Sensing of the Responses of Soybean Plants to Environmental Stresses 243

The effect of combined stress action of salinity + UV-B radiation was assessed by biochemical parameters: phenols, proline, malondialdehyde, and hydrogen peroxide. The

The content of phenols decreased with 34.2% and 17% for low and high NaCl concentration, respectively. This is an indicator for decreasing of the action of salinity stress on the plants from the group treated with 80 mM NaCl +UV-B. The production of the MDA is a very sensitive stress marker. When its content is decreased it is a sign of the induction of sustainability to the applied stress. The increase in H2O2 and MDA contents was shown to be related to the amount of stress and well correlated with lipid membrane damage (Demiral & Turkan, 2005). In our experiment the amount of MDA increased for 40 mM NaCl + UV-B treatment with 4.5% and decreased in the other case with 7.6%. The hydrogen peroxide levels indicate that under the action of salinity stress + UV-B radiation the H2O2 content becomes higher with the increasing NaCl concentration. Hydrogen peroxide is known to damage cellular components and provoke structural modifications of proteins and lipids (Mandhania et al., 2006). Our experiment shows that a considerable increase of proline content takes place under the influence of salinity + UV-B at the lower concentration. By contrast, at 80 mM NaCl a significant decrease of proline by about 10.4%

contents of the evaluated stress markers are shown in Table 13.

was observed.

fixing soybean plants.

H2O2.

Stress Markers, Pigments

Phenols

Proline

MDA

H2O2

Control +UV-B

(μM/g DW) 5.173 ±0.07 3.442±0.9

(nmol/g DW) 8.942±0.87 22.955±1.9

(nmol/g DW) 24.68±0.3 25.78±0.82

(μM/g DW) 8.752±0.06 20.528±0.04

Table 13. Values of the biochemical parameters for the combined treatment of nitrogen

Spectral data analysis revealed that in the case of the action of a single salinity stress on the young nitrogen fixing soybean plants there were statistically significant differences between the reflectance spectra of the leaves of control and treated plants at the two NaCl concentrations in all of the ranges examined with the exception of two wavelengths in NIR at 40 mM NaCl concentration and one wavelength at 80 mM NaCl concentration. In the SRC this effect was manifested by decreasing of the values in the green and red ranges and increasing in NIR. The results indicated that the plants were under conditions of stress which has been better pronounced for the higher NaCl concentration where necrotic and chlorotic lesions on the leaves have appeared due to chlorophyll degradation. This finding was established by evaluated biochemical markers of stress: phenols, proline, MDA, and

In the case of treatment with (40 mm NaCl + UV-B) and (80 mm NaCl + UV-B) the results indicated that stress in the plants was also present but the influence of UV-B radiation after salinity reduces the consequences of salinity stress especially at the higher NaCl concentration. In the spectral reflectance characteristics this effect is manifested through an

40 mM NaCl + UV-B

34.2%

156.7%

4.5%

134.5%

80 mM NaCl +UV-B

4.289 ±0.04 17%

8.017±0.66 10.4%

22.80±0.66 7.6%

50.903±0.02 481.6%

The results from the statistical analysis concerning the combined impact of stresses to soybean plants are set out in Table 12. They indicate the increasing number of wavelengths in which the differences of the SRC against the control are non-significant. In the case of (40 mM NaCl + UV-B) the differences are non-significant in the whole NIR range. For (80 mM NaCl + UV-B) treatment they are non-significant in four of the wavelengths.

Fig. 17. Averaged spectral reflectance characteristics of control and treated nitrogen fixing soybean plants with: 40 mM NaCl + UV-B and 80 mM NaCl + UV-B.


Table 12. Significance p-level of the t-criterion in the cases of (40 mM NaCl +UV-B) and (80 mM NaCl + UV-B) treatment.

The results from the statistical analysis concerning the combined impact of stresses to soybean plants are set out in Table 12. They indicate the increasing number of wavelengths in which the differences of the SRC against the control are non-significant. In the case of (40 mM NaCl + UV-B) the differences are non-significant in the whole NIR range. For (80 mM

> 450 550 650 750 850 Wavelength, nm

Fig. 17. Averaged spectral reflectance characteristics of control and treated nitrogen fixing

Pairs Control 40 mM NaCl +UV-B 80 mM NaCl + UV-B compared mean *pSt* mean *pSt* mean λ1/λ1c 11.03 <0.001 10.38 <0.001 9.92 λ2/λ2c 14.63 <0.001 13.60 <0.001 13.06 λ3/λ3c 15.47 <0.001 14.30 <0.001 13.80 λ4/λ4c 4.41 <0.001 3.99 0.541 4.34 λ5/λ5c 19.79 0.006 19.71 <0.001 17.08 λ6/λ6c 33.09 0.733 33.37 0.067 32.23 λ7/λ7c 35.99 0.344 36.87 0.835 36.09 λ8/λ8c 43.22 0.214 44.80 0.102 45.29 Table 12. Significance p-level of the t-criterion in the cases of (40 mM NaCl +UV-B) and (80

soybean plants with: 40 mM NaCl + UV-B and 80 mM NaCl + UV-B.

NaCl + UV-B) treatment they are non-significant in four of the wavelengths.

Control+UV 40mM+UV\_Nfix 80mM+UV\_Nfix

0

mM NaCl + UV-B) treatment.

10

20

30

Spectral reflectance, %

40

50

The effect of combined stress action of salinity + UV-B radiation was assessed by biochemical parameters: phenols, proline, malondialdehyde, and hydrogen peroxide. The contents of the evaluated stress markers are shown in Table 13.

The content of phenols decreased with 34.2% and 17% for low and high NaCl concentration, respectively. This is an indicator for decreasing of the action of salinity stress on the plants from the group treated with 80 mM NaCl +UV-B. The production of the MDA is a very sensitive stress marker. When its content is decreased it is a sign of the induction of sustainability to the applied stress. The increase in H2O2 and MDA contents was shown to be related to the amount of stress and well correlated with lipid membrane damage (Demiral & Turkan, 2005). In our experiment the amount of MDA increased for 40 mM NaCl + UV-B treatment with 4.5% and decreased in the other case with 7.6%. The hydrogen peroxide levels indicate that under the action of salinity stress + UV-B radiation the H2O2 content becomes higher with the increasing NaCl concentration. Hydrogen peroxide is known to damage cellular components and provoke structural modifications of proteins and lipids (Mandhania et al., 2006). Our experiment shows that a considerable increase of proline content takes place under the influence of salinity + UV-B at the lower concentration. By contrast, at 80 mM NaCl a significant decrease of proline by about 10.4% was observed.


Table 13. Values of the biochemical parameters for the combined treatment of nitrogen fixing soybean plants.

Spectral data analysis revealed that in the case of the action of a single salinity stress on the young nitrogen fixing soybean plants there were statistically significant differences between the reflectance spectra of the leaves of control and treated plants at the two NaCl concentrations in all of the ranges examined with the exception of two wavelengths in NIR at 40 mM NaCl concentration and one wavelength at 80 mM NaCl concentration. In the SRC this effect was manifested by decreasing of the values in the green and red ranges and increasing in NIR. The results indicated that the plants were under conditions of stress which has been better pronounced for the higher NaCl concentration where necrotic and chlorotic lesions on the leaves have appeared due to chlorophyll degradation. This finding was established by evaluated biochemical markers of stress: phenols, proline, MDA, and H2O2.

In the case of treatment with (40 mm NaCl + UV-B) and (80 mm NaCl + UV-B) the results indicated that stress in the plants was also present but the influence of UV-B radiation after salinity reduces the consequences of salinity stress especially at the higher NaCl concentration. In the spectral reflectance characteristics this effect is manifested through an

Spectral Remote Sensing of the Responses of Soybean Plants to Environmental Stresses 245

The averaged normalized fluorescence spectra of the nitrogen fixing soybean plants subjected to combined stress factors (salinity + UV-B radiation) for the two NaCl concentrations are displayed in Fig. 19. It is observed that the differences between the spectra are less pronounced than those at single salinity stress impact. For the combined action of (40 mM NaCl+UV-B) the averaged spectrum has higher values than control due to induced tolerance to salinity stress from the action of UV-B radiation. For (80 mM NaCl+UV-B) treatment the values are lower and more close-set to the control due to the

> Control+UV-B 80 mM NaCl+UV-B 40 mM NaCl+UV-B

positive action of UV-B radiation expressed through the decreasing effect of salinity.

600 650 700 750 800 850 900 Wavelength, nmn

Fig. 19. Normalized average fluorescence spectra of control and treated with (40 mM NaCl +

Table 15 shows the results from the Student's t-criterion for the combined stress treatment of the soybean plants. The differences against the control spectra are statistically significant at

The results revealed that the salinity treatment at two NaCl concentrations significantly changed the fluorescence spectra of the nitrogen fixing soybean plants in the spectral range spanning from arising forefront to the second maximum (640-740 nm) due to salinity stress caused in the plants. In the case of initial salinity treatment of the plants followed by their irradiation with UV-B light the differences between the fluorescence spectra decreased due to the favourable effect of the UV-B light. These results were in compliance with the findings concerning the leaf spectral reflectance and biochemical parameters measured on the same nitrogen fixing soybean plants, treated with 40 mM and 80 mM NaCl concentrations and

UV-B) and (80 mM NaCl + UV-B) nitrogen fixing soybean plants.

two of the investigated wavelengths for the two cases.

0

salinity + UV-B radiation.

0.2

0.4

0.6

Fluorescence, relative units

0.8

1

1.2

increase of SRC values for treated plants and their approaching to the control SRC. This finding was established by the analysis of the evaluated biochemical stress markers.

The averaged fluorescence spectra of leaves of the same nitrogen fixing soybean plants treated with the two NaCl concentrations are displayed in Fig. 18 (Krezhova et al., 2011b). All spectra are normalized to their second maximum, which in this case is at λ4=λm= 738 nm. Changes in the spectra of treated plants (second and third groups) against the control (first group) increased with increasing NaCl amount and were observed in the spectral range spanning from arising forefront to the second maximum (640-740 nm). At their rear slope, the fluorescence spectra were with almost equal values.

Fig. 18. Normalized average fluorescence spectra of control and treated with 40 mM NaCl and 80 mM NaCl nitrogen fixing soybean plants.

The results from the statistical analysis are presented in Table 14. Statistically significant differences were evaluated by Student's t-criterion at p<0.05 for the data at first three wavelengths for the two NaCl concentrations.


Table 14. Significance p-level of the t-criterion in the cases of 40 mM NaCl and 80 mM NaCl salinity of nitrogen fixing soybean plants.

increase of SRC values for treated plants and their approaching to the control SRC. This

The averaged fluorescence spectra of leaves of the same nitrogen fixing soybean plants treated with the two NaCl concentrations are displayed in Fig. 18 (Krezhova et al., 2011b). All spectra are normalized to their second maximum, which in this case is at λ4=λm= 738 nm. Changes in the spectra of treated plants (second and third groups) against the control (first group) increased with increasing NaCl amount and were observed in the spectral range spanning from arising forefront to the second maximum (640-740 nm). At their rear slope,

> control 40 mM NaCl 80 mM NaCl

600 650 700 750 800 850 900 Wavelength, nm

Fig. 18. Normalized average fluorescence spectra of control and treated with 40 mM NaCl

The results from the statistical analysis are presented in Table 14. Statistically significant differences were evaluated by Student's t-criterion at p<0.05 for the data at first three

Pairs Control 40 mM NaCl 80 mM NaCl compared mean *pSt* mean *pSt* mean λ1/λ1c 0.335 <0.001 0.565 <0.001 0.221 λ2/λ2c 1.122 <0.001 0.770 <0.001 0.613 λ3/λ3c 0.912 <0.001 0.803 <0.001 0.735 λ5/λ5c 0.610 0.987 0.630 0.874 0.610 Table 14. Significance p-level of the t-criterion in the cases of 40 mM NaCl and 80 mM NaCl

finding was established by the analysis of the evaluated biochemical stress markers.

the fluorescence spectra were with almost equal values.

0

and 80 mM NaCl nitrogen fixing soybean plants.

wavelengths for the two NaCl concentrations.

salinity of nitrogen fixing soybean plants.

0.2

0.4

0.6

Fluorescence, relative units

0.8

1

1.2

The averaged normalized fluorescence spectra of the nitrogen fixing soybean plants subjected to combined stress factors (salinity + UV-B radiation) for the two NaCl concentrations are displayed in Fig. 19. It is observed that the differences between the spectra are less pronounced than those at single salinity stress impact. For the combined action of (40 mM NaCl+UV-B) the averaged spectrum has higher values than control due to induced tolerance to salinity stress from the action of UV-B radiation. For (80 mM NaCl+UV-B) treatment the values are lower and more close-set to the control due to the positive action of UV-B radiation expressed through the decreasing effect of salinity.

Fig. 19. Normalized average fluorescence spectra of control and treated with (40 mM NaCl + UV-B) and (80 mM NaCl + UV-B) nitrogen fixing soybean plants.

Table 15 shows the results from the Student's t-criterion for the combined stress treatment of the soybean plants. The differences against the control spectra are statistically significant at two of the investigated wavelengths for the two cases.

The results revealed that the salinity treatment at two NaCl concentrations significantly changed the fluorescence spectra of the nitrogen fixing soybean plants in the spectral range spanning from arising forefront to the second maximum (640-740 nm) due to salinity stress caused in the plants. In the case of initial salinity treatment of the plants followed by their irradiation with UV-B light the differences between the fluorescence spectra decreased due to the favourable effect of the UV-B light. These results were in compliance with the findings concerning the leaf spectral reflectance and biochemical parameters measured on the same nitrogen fixing soybean plants, treated with 40 mM and 80 mM NaCl concentrations and salinity + UV-B radiation.

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Table 15. Significance p-level of the t-criterion in the cases of 40 mM NaCl+UV-B and 80 mM NaCl+UV-B treatment for nitrogen fixing soybean plants.
