**6.1 Influence of a single stress factor at soybean plants**

The effect of salinity on leaf spectral reflectance of soybean plants for the first part of the experiments (see Fig. 3) is shown in Fig. 12 (Krezhova et al., 2009a). The SRC were averaged over all studied areas (pixels) of leaves of control and treated with each of the two salt concentrations plants. The discrepancy (lack of coincidence or very small differences) between the characteristics of control and treated by 40 mM NaCl plants was observed in the green (520-580 nm, maximal chlorophyll reflection) and NIR ranges.

At 80 mM NaCl the values of the average SRC of treated leaves with respect to control decrease significantly in both the green and red (640-680 nm) ranges. In the red edge (680- 720 nm) it is observed a shift to longer wavelengths (8 nm) indicating the occurrence of stress. Necrosis spots were seen on some of the leaves of plants treated with this NaCl concentration (Fig. 3 c). The red edge position changed significantly with the increase in NaCl concentration applied to the plants and it is a consequence of the decreased chlorophyll content determined by biochemical method. In the NIR range the reflectance changed non-significant against the control.

Spectral Remote Sensing of the Responses of Soybean Plants to Environmental Stresses 233

Similar results are obtained through linear DA. The grouping variable used on the first stage of DA implementation was designed to consist of only two groups - control and by one of the treated with different NaCl concentration plants. Since the DA significant p-level for the case of 40 mM NaCl turned out to be >>0.05 with all wavelengths (0.12<p<0.98) the *pDA* are not shown in the Table 2. Anyway, if for example the three dimensional space (λ4, λ6, λ7) is

Discriminant analysis, 80 mM NaCl

Number of incorrectly classified cases

*pDA*

λ1 <0.001 0 λ2 <0.001 0 λ3 <0.001 0 λ<sup>4</sup> <0.001 18 from 69 λ<sup>5</sup> <0.001 1 from 69 λ<sup>6</sup> <0.001 6 from 69 λ<sup>7</sup> <0.001 11 from 69 λ<sup>8</sup> 0.95 29 from 69

Table 2. Significance p-level of the Discriminant analysis model in the case of 80 mM NaCl

The contents of the evaluated stress markers and chlorophyll a and b are shown in Table 3. The values of control plants were taken as 100%. The 40 mM NaCl treatment did not provoke changes in the phenol content in soybean leaves, while 80 mM NaCl caused a decrease of the content by about 33.6%. Such sharp decrease of phenols content in the leaves treated by 80 mM NaCl gives grounds to consider the phenols as playing the role of

NaCl

2%

12.3%

18%

25%

42%

21%

23%

23%

80 mM NaCl

81.8±0.9 33.6%

48.1±1.9 56.2

140.2±5.6 44%

6.3±0.02 7%

4.23±0.03 70%

0.71±0.03 49%

0.35±0.01 42%

> 76.3 46%

pigments Control 40 mM

(μM/gDW) 123.2 ±2.1 122.9 ±1.1

(nmol/gDW) 30.8±0.7 34.6±2.3

(nmol/g DW) 97.6±5.3 114.9±6.8

(μM/gDW) 6.8±0.06 5.1±0.04

(μM/gDW) 2.49±0.01 3.59±0.04

(mg/g FW) 1.39±0.03 1.09±0.02

(mg/g FW) 0.60±0.01 0.46±0.03

(%) 52.4 64.1

Table 3. Values of the biochemical parameters of salinity treated soybean plants.

used, the *pDA* level is p<0.001 while the incorrectly classified cases are 12 from 69.

One dimensional spaces

salinity.

endogen antioxidant in plants.

Stress markers,

Phenols,

Proline,

MDA

H2O2

Thiol groups

Chlorophyll а

Chlorophyll b

GSSG/ TG

Fig. 12. Averaged spectral reflectance characteristics of control and treated with 40 mM NaCl and 80 mM NaCl soybean plants.

The results (p-level of the difference between SRC means of treated plants and SRC means control plants at a given λ) from the statistical analysis of the spectral data are set out in Table 1 and Table 2. In Table 1 *pst* stands for the significance p-level of the Student's tcriterion. In Table 2 *pDA* designates the significance p-level of the DA model. The index *c* stands for reflectance or fluorescence data of control plants. Statistically significant differences between SRC means of control and treated at 80 mM NaCl concentration were detected at p<0.05 by means of the Student's t-criterion in each wavelength with the exception of λ8 in the NIR range. The impact of 40 mM NaCl salinity is not sufficient to provoke detectable changes in SRC in the wavelengths examined.


Table 1. Significance p-level of the Student's t-criterion in the cases of 40 mM NaCl and 80 mM NaCl salinity.

Control 40 mM NaCl 80 mM NaCl

450 550 650 750 850 Wavelength, nm

Fig. 12. Averaged spectral reflectance characteristics of control and treated with 40 mM

The results (p-level of the difference between SRC means of treated plants and SRC means control plants at a given λ) from the statistical analysis of the spectral data are set out in Table 1 and Table 2. In Table 1 *pst* stands for the significance p-level of the Student's tcriterion. In Table 2 *pDA* designates the significance p-level of the DA model. The index *c* stands for reflectance or fluorescence data of control plants. Statistically significant differences between SRC means of control and treated at 80 mM NaCl concentration were detected at p<0.05 by means of the Student's t-criterion in each wavelength with the exception of λ8 in the NIR range. The impact of 40 mM NaCl salinity is not sufficient to

> Student's t-criterion 40 mM NaCl 80 mM NaCl Pairs compared *pst* Pairs compared *pst*

λ1/λ1c 0.328 λ1/λ1c <0.001 λ2/λ2c 0.210 λ2/λ2c <0.001 λ3/λ3c 0.185 λ3/λ3c <0.001 λ4/λ4c 0.061 λ4/λ4c <0.001 λ5/λ5c 0.125 λ5/λ5c <0.001 λ6/λ6c 0.120 λ6/λ6c <0.001 λ7/λ7c 0.082 λ7/λ7c <0.001 λ8/λ8c 0.285 λ8/λ8c 0.94 Table 1. Significance p-level of the Student's t-criterion in the cases of 40 mM NaCl and 80

0

provoke detectable changes in SRC in the wavelengths examined.

NaCl and 80 mM NaCl soybean plants.

mM NaCl salinity.

10

20

30

Reflectance, %

40

50

60

Similar results are obtained through linear DA. The grouping variable used on the first stage of DA implementation was designed to consist of only two groups - control and by one of the treated with different NaCl concentration plants. Since the DA significant p-level for the case of 40 mM NaCl turned out to be >>0.05 with all wavelengths (0.12<p<0.98) the *pDA* are not shown in the Table 2. Anyway, if for example the three dimensional space (λ4, λ6, λ7) is used, the *pDA* level is p<0.001 while the incorrectly classified cases are 12 from 69.


Table 2. Significance p-level of the Discriminant analysis model in the case of 80 mM NaCl salinity.

The contents of the evaluated stress markers and chlorophyll a and b are shown in Table 3. The values of control plants were taken as 100%. The 40 mM NaCl treatment did not provoke changes in the phenol content in soybean leaves, while 80 mM NaCl caused a decrease of the content by about 33.6%. Such sharp decrease of phenols content in the leaves treated by 80 mM NaCl gives grounds to consider the phenols as playing the role of endogen antioxidant in plants.


Table 3. Values of the biochemical parameters of salinity treated soybean plants.

Spectral Remote Sensing of the Responses of Soybean Plants to Environmental Stresses 235

**1 - Control 2 - 40mM NaCl 3 - 80mM NaCl**

**1**

**3**

**2**

600 650 700 750 800 850 900 Wavelength, nm

Fig. 13. Averaged fluorescence spectra of control and treated with: 40 mM NaCl and 80 mM

Student's t-criterion Salinity 40 mM NaCl Salinity 80 mM NaCl Pairs compared *st p* Pairs compared *st p*

0.202 51 51 *<sup>c</sup>*

Table 4. Significance p-level of the t-criterion for the set of fluorescence indices.

the fluorescence spectra due to caused salinity stress in the soybean plants.

 1 1 

 5 5 

> 

 1 1 *m mc* 

*S Sc* 0.012 *S Sc* <0.001

Similar results are obtained through linear discriminant analysis by making use of one dimensional spaces defined by each of the indices herein used, the *pDA* level is p<0.05 while the incorrectly classified cases are not more than 5 cases from 24. The two indices λm and S also indicated perspective possibilities for detection of salinity injures on soybean plants. The results from the chlorophyll fluorescence analysis revealed that the low NaCl concentration applied does not produce statistically significant changes in the leaf fluorescence. Applying of high NaCl concentration lead to significantly changed forefront of

In summary, the results from the first part of experiments have shown that there is a difference in the spectral reflectance characteristics in response to different salt

*<sup>c</sup>* <0.001

0.007

*<sup>c</sup>* 0.009

<0.001

0.0

*<sup>c</sup>* 0.955

0.045

*<sup>c</sup>* 0.257

NaCl soybean leaves.

 1 1 

 5 5 

51 51 *<sup>c</sup>*

 

 1 1 *m mc* 

0.2

0.4

0.6

Relative emission intensity

0.8

1.0

A number of authors have observed that most plant species exhibit a remarkable increase in their proline content in consequence of the action of different kinds of stress such as UVradiation, drought, salinity, etc. (Sivakumar et al., 2000; Jogeswar et al., 2006; Sun et al., 2008). Characteristic changes in proline content at the salinity stress are described in roots and leaves of alfalfa and pea (Tramontano & Jouve, 1997), and in leaves of cotton and bean (Brankova et al., 2005). Our results show that an increase of proline content by about 12% takes place under the influence of salinity at the lower concentration. A more significant increase of proline by about 56% was observed at 80 mM NaCl concentration.

An increase in hydrogen peroxide and MDA contents upon salt stress has been reported for different plant spices (Yang et al., 2008). This increase was shown to be related to the amount of stress and well correlated with lipid membrane damage. Our results show that salinity at 40 mM NaCl leads to an increase of MDA by about 18% in comparison with the control. A more substantial increase is observed at 80 mM NaCl that reaches 44%. These results agree with the findings of Jogeswar et al. (2006) who have established a significant increase of MDA at treatment of sorghum (Sorghum bicolour) with 150 mM NaCl.

Unexpectedly, in our experiments the Н2О2 content was found lower by about 25% at the low salinity and by about 7% at the high salinity. This finding provides grounds to continue our investigations in order to determine the activity of enzymes from the antioxidant system (catalase and peroxidase) using Н2О2 as substrate.

The measurements of thiol groups observed an increase by about 40% at the treatment with 40 mM NaCl. The salinity at 80 mM NaCl lead to about doubling (by 70%) of the free -SH groups. By our opinion, the increase of thiol groups might serve as a marker of damages induced by salinity stress. The ratio of oxidized form glutathione to total glutathione (GSSG/TG) is much higher than the control at 80 mM NaCl which is evidence of a strong reduction of the capacity of antioxidant system for the plants under study.

The averaged fluorescence spectra over 20 leaves of the control and by 20 treated leaves with each of the two NaCl concentrations of the same soybean plants used in the first part of the experiments are shown in Fig. 13 (Iliev et al., 2009a). All spectra are normalized to their second maximum at λm which in this case coincided with the wavelength of 738 nm. Changes in the fluorescence spectra of treated plants against the control were predominantly observed in the arising forefront. Curve 2 (the averaged leaf spectrum of plants treated with 40 mM NaCl) slightly differs against control curve 1 in the spectral range 640-680 nm. Curve 3 (80 mM NaCl treatment) differs against curve 1 significantly within the spectral range 600-740 nm.

The Student's t-criterion and linear DA were applied to estimate the statistical significance of the differences between the means of the indices chosen to characterize the fluorescence spectrum (halfwidth, wavelength at the first maximum, area) defined as, (see Fig. 11):



The main results of the statistical analysis are summarized in Table 4 and Table 5. It is seen in Table 4 that the changes of the indices under the conditions of 80 mM NaCl concentration could be detected at p<0.05 by means of each of the indices. Also, it is clear that the impact of 40 mM NaCl is not sufficient to induce detectable changes in any of the fluorescence indices.

A number of authors have observed that most plant species exhibit a remarkable increase in their proline content in consequence of the action of different kinds of stress such as UVradiation, drought, salinity, etc. (Sivakumar et al., 2000; Jogeswar et al., 2006; Sun et al., 2008). Characteristic changes in proline content at the salinity stress are described in roots and leaves of alfalfa and pea (Tramontano & Jouve, 1997), and in leaves of cotton and bean (Brankova et al., 2005). Our results show that an increase of proline content by about 12% takes place under the influence of salinity at the lower concentration. A more significant

An increase in hydrogen peroxide and MDA contents upon salt stress has been reported for different plant spices (Yang et al., 2008). This increase was shown to be related to the amount of stress and well correlated with lipid membrane damage. Our results show that salinity at 40 mM NaCl leads to an increase of MDA by about 18% in comparison with the control. A more substantial increase is observed at 80 mM NaCl that reaches 44%. These results agree with the findings of Jogeswar et al. (2006) who have established a significant

Unexpectedly, in our experiments the Н2О2 content was found lower by about 25% at the low salinity and by about 7% at the high salinity. This finding provides grounds to continue our investigations in order to determine the activity of enzymes from the antioxidant system

The measurements of thiol groups observed an increase by about 40% at the treatment with 40 mM NaCl. The salinity at 80 mM NaCl lead to about doubling (by 70%) of the free -SH groups. By our opinion, the increase of thiol groups might serve as a marker of damages induced by salinity stress. The ratio of oxidized form glutathione to total glutathione (GSSG/TG) is much higher than the control at 80 mM NaCl which is evidence of a strong

The averaged fluorescence spectra over 20 leaves of the control and by 20 treated leaves with each of the two NaCl concentrations of the same soybean plants used in the first part of the experiments are shown in Fig. 13 (Iliev et al., 2009a). All spectra are normalized to their second maximum at λm which in this case coincided with the wavelength of 738 nm. Changes in the fluorescence spectra of treated plants against the control were predominantly observed in the arising forefront. Curve 2 (the averaged leaf spectrum of plants treated with 40 mM NaCl) slightly differs against control curve 1 in the spectral range 640-680 nm. Curve 3 (80 mM NaCl treatment) differs against curve 1 significantly within the

The Student's t-criterion and linear DA were applied to estimate the statistical significance of the differences between the means of the indices chosen to characterize the fluorescence spectrum (halfwidth, wavelength at the first maximum, area) defined as, (see Fig. 11): - halfwidth of the fluorescence spectrum (λ5 - λ1); λ1 (relative emission intensity, REI = 0.5

The main results of the statistical analysis are summarized in Table 4 and Table 5. It is seen in Table 4 that the changes of the indices under the conditions of 80 mM NaCl concentration could be detected at p<0.05 by means of each of the indices. Also, it is clear that the impact of 40 mM NaCl is not sufficient to induce detectable changes in any of the fluorescence


increase of proline by about 56% was observed at 80 mM NaCl concentration.

increase of MDA at treatment of sorghum (Sorghum bicolour) with 150 mM NaCl.

reduction of the capacity of antioxidant system for the plants under study.

in the forefront) and λ5 (REI = 0.5 in the rear slope);

(catalase and peroxidase) using Н2О2 as substrate.

spectral range 600-740 nm.

indices.

Fig. 13. Averaged fluorescence spectra of control and treated with: 40 mM NaCl and 80 mM NaCl soybean leaves.


Table 4. Significance p-level of the t-criterion for the set of fluorescence indices.

Similar results are obtained through linear discriminant analysis by making use of one dimensional spaces defined by each of the indices herein used, the *pDA* level is p<0.05 while the incorrectly classified cases are not more than 5 cases from 24. The two indices λm and S also indicated perspective possibilities for detection of salinity injures on soybean plants.

The results from the chlorophyll fluorescence analysis revealed that the low NaCl concentration applied does not produce statistically significant changes in the leaf fluorescence. Applying of high NaCl concentration lead to significantly changed forefront of the fluorescence spectra due to caused salinity stress in the soybean plants.

In summary, the results from the first part of experiments have shown that there is a difference in the spectral reflectance characteristics in response to different salt

Spectral Remote Sensing of the Responses of Soybean Plants to Environmental Stresses 237

The second part of experiments was aimed at studying of the salinity effect on nitrogen fixing soybean plants. The measurements were performed on 25 areas (pixels) of randomly picked off leaves from each group of plants. The averaged SRC are displayed in Fig. 14 (Krezhova et al., 2009b). It is seen that the values of the SRC of the treated leaves with respect to control decrease significantly in both the green and red (450-680 nm) spectral ranges. In the red edge region it is observed a shift to longer wavelengths in correspondence with the increase in NaCl concentration applied, which is indicating occurrence of plant stress. For the case of 40 mM NaCl the shift is 2 nm while for the high salt concentration it is 6 nm. In the NIR range, the reflectance at low NaCl concentration increased while at high NaCl concentration it decreased due to the changes of water and nitrogen content in the

The results of application of the Student's t-criterion are presented in Table 6. Statistically significant are differences for which p<0.05 and only the differences in wavelengths λ7 and

40 mM NaCl 80 mM NaCl Control

compared *pst* mean *pst* mean mean λ1/λ1c <0.001 14.41 <0.001 12.10 15.47 λ2/λ2c <0.001 18.96 <0.001 16.13 20.74 λ3/λ3c <0.001 19.86 <0.001 16.93 21.74 λ4/λ4c 0.0027 5.46 <0.001 4.87 5.92 λ5/λ5c <0.001 22.95 <0.001 20.76 25.22 λ6/λ6c 0.0298 44.98 <0.001 41.15 46.32 λ7/λ7c 0.3117 50.50 <0.001 46.27 51.15 λ8/λ8c 0.3048 62.61 0.001 57.98 61.76 Table 6. Significance p-level of the t-criterion in the case of 40 mM NaCl and 80 mM NaCl

Linear DA was implemented making use of one-dimensional spaces defined by each one of the wavelengths. Table 7 shows that probability levels *pDA* coincide with that of the Student's t-criterion as the grouping variable consisted for each concentration of only two classes: control and treated plants (40 mM NaCl or 80 mM NaCl) at a given wavelength. At λ7 and λ8 (the same as for the t-criterion) statistically significant differences between SRC means for control and tread plants were not observed. Therefore the number of incorrectly

To illustrate better discriminative DA possibilities we performed DA in a two-dimensional space defined by the wavelengths λ7 and λ8, which manifested worst results when applied separately. Making use of data for concentration 40mM NaCl the p-level turned out to be

The contents of the evaluated biochemical parameters - stress markers (phenols, proline, MDA, H2O2, free thiol groups, ratio of oxidized to total glutathione GSSG/TG) and content of chlorophyll a and b, are shown in Table 8. The values for control plants were taken as 100%. The 40 mM NaCl concentration lead to a phenol content decrease of about 19%, whereas the 80 mM NaCl salinity treatment provoked their much stronger decrease (by 59%). At 40 mM NaCl the proline content increased with 8%. A considerable increase of the proline (73%) was observed at 80 mM NaCl concentration. Under salinity stress most plant

leaves.

salinity.

λ8 at 40 mM NaCl salinity are non-significant.

classified cases was maximal at these wavelengths.

<0.001 and the number of incorrectly classified cases was only 12.

Pairs

concentration treatment of soybean plants. The shift of the red edge position correlated with increased concentration of the salinity. Low NaCl concentration (40 mM) caused insignificant changes in the SRC and led to salinity tolerance whereas high NaCl concentration (80 mM) induced considerable SRC changes implying presence of salinity stress in soybean plants. This finding was in agreement with the outcome from the chlorophyll fluorescence analysis carried out on the same plants and evaluated biochemical stress markers such as phenols, proline, malondialdehyde, thiol groups, hydrogen peroxide, and leaf pigment contents (Chl a and Chl b).


Table 5. Significance p-level of the Discriminant analysis model for the set of fluorescence indices.

Fig. 14. Averaged spectral reflectance characteristics of control and treated with 40 mM and 80 mM NaCl nitrogen fixing soybean plants.

concentration treatment of soybean plants. The shift of the red edge position correlated with increased concentration of the salinity. Low NaCl concentration (40 mM) caused insignificant changes in the SRC and led to salinity tolerance whereas high NaCl concentration (80 mM) induced considerable SRC changes implying presence of salinity stress in soybean plants. This finding was in agreement with the outcome from the chlorophyll fluorescence analysis carried out on the same plants and evaluated biochemical stress markers such as phenols, proline, malondialdehyde, thiol groups, hydrogen peroxide,

Discriminant Analysis

Number of incorrectly classified cases

<sup>1</sup> 0.996 <0.001 0

*S* 0.0123 8/22 <0.001 0 Table 5. Significance p-level of the Discriminant analysis model for the set of fluorescence

> NaCl 40mM NaCl 80 mM Control

<sup>1</sup>*<sup>m</sup>* 0.044 5/22 0.0132 5 from 24

<sup>5</sup> 0.256 0.019 3 from 24

0.199 <0.001 1 from 24

450 550 650 750 850 Wavelength, nm

Fig. 14. Averaged spectral reflectance characteristics of control and treated with 40 mM and

Salinity 40 mM NaCl Salinity 80 mM NaCl

*DA p*

Number of incorrectly classified cases

and leaf pigment contents (Chl a and Chl b).

*DA p*

One dimensional spaces

 5 1 

0

80 mM NaCl nitrogen fixing soybean plants.

10

20

30

40

Reflectance, relative units

50

60

70

indices.

The second part of experiments was aimed at studying of the salinity effect on nitrogen fixing soybean plants. The measurements were performed on 25 areas (pixels) of randomly picked off leaves from each group of plants. The averaged SRC are displayed in Fig. 14 (Krezhova et al., 2009b). It is seen that the values of the SRC of the treated leaves with respect to control decrease significantly in both the green and red (450-680 nm) spectral ranges. In the red edge region it is observed a shift to longer wavelengths in correspondence with the increase in NaCl concentration applied, which is indicating occurrence of plant stress. For the case of 40 mM NaCl the shift is 2 nm while for the high salt concentration it is 6 nm. In the NIR range, the reflectance at low NaCl concentration increased while at high NaCl concentration it decreased due to the changes of water and nitrogen content in the leaves.

The results of application of the Student's t-criterion are presented in Table 6. Statistically significant are differences for which p<0.05 and only the differences in wavelengths λ7 and λ8 at 40 mM NaCl salinity are non-significant.


Table 6. Significance p-level of the t-criterion in the case of 40 mM NaCl and 80 mM NaCl salinity.

Linear DA was implemented making use of one-dimensional spaces defined by each one of the wavelengths. Table 7 shows that probability levels *pDA* coincide with that of the Student's t-criterion as the grouping variable consisted for each concentration of only two classes: control and treated plants (40 mM NaCl or 80 mM NaCl) at a given wavelength. At λ7 and λ8 (the same as for the t-criterion) statistically significant differences between SRC means for control and tread plants were not observed. Therefore the number of incorrectly classified cases was maximal at these wavelengths.

To illustrate better discriminative DA possibilities we performed DA in a two-dimensional space defined by the wavelengths λ7 and λ8, which manifested worst results when applied separately. Making use of data for concentration 40mM NaCl the p-level turned out to be <0.001 and the number of incorrectly classified cases was only 12.

The contents of the evaluated biochemical parameters - stress markers (phenols, proline, MDA, H2O2, free thiol groups, ratio of oxidized to total glutathione GSSG/TG) and content of chlorophyll a and b, are shown in Table 8. The values for control plants were taken as 100%. The 40 mM NaCl concentration lead to a phenol content decrease of about 19%, whereas the 80 mM NaCl salinity treatment provoked their much stronger decrease (by 59%). At 40 mM NaCl the proline content increased with 8%. A considerable increase of the proline (73%) was observed at 80 mM NaCl concentration. Under salinity stress most plant

Spectral Remote Sensing of the Responses of Soybean Plants to Environmental Stresses 239

bring to salinity stress in the nitrogen fixing soybean plants and to decline of the biological nitrogen fixation. The red edge shift to longer wavelengths is an indicator of stress and is

The course of the averaged fluorescence spectra over 20 control and 20 salinity treated leaves of nitrogen fixing soybean plants used for the second part of the experiments is shown in Fig. 15 (Iliev et al., 2009b). All spectra were normalized against their second maximum. Changes in the spectra of treated leaves against the controls were significant in the forefront and in the spectral range between first and second maximums (680-740 nm).

**1**

**1 - Control plants 2 - 40 mM NaCl 3 - 80 mM NaCl**

**2 3**

600 650 700 750 800 850 900 Wavelength, nm

> 80 mM NaCl

80 mM

NaCl Control

Fig. 15. Averaged fluorescence spectra of control and treated with 40 mM and 80 mM NaCl

The results of the Student's t-criterion and linear DA are displayed in Table 9 and Table 10. For analysis, five fluorescence values for each spectrum (from all 910) in characteristic wavelengths in the spectral range 600-900 nm were selected: 1 (at the middle of the forefront), 2 (first maximum), 3 (at the middle between first and second maximum), <sup>4</sup>

compared *pst* mean *pst* mean mean λ1/λ1c <0.001 911.6 <0.001 758.1 1345.8 λ2/λ2c <0.001 2100.6 <0.001 1878.4 2672.0 λ3/λ3c 0.0016 2097.0 <0.001 1994.9 2332.9 λ4/λ4c 0.318 2436.9 0.2305 2408.1 2506.8 λ5/λ5c 0.480 1494.2 0.4919 1490.1 1525.1 Table 9. Significance p-level of the t-criterion for the set of amplitudes of the fluorescence

correlated with the decreased chlorophyll content and salinity rate.

0.0

(second maximum) and 5 (at the middle of rear slope), see Fig. 11.

40 mM NaCl

nitrogen fixing soybean plants.

Pairs 40 mM

spectra.

NaCl

0.4

0.8

Relative emission intensity

1.2


Table 7. Significance p-level of the linear DA in the case of 40 mM NaCl and 80 mM NaCl salinity of nitrogen fixing soybean plants.

species exhibit a remarkable increase in the proline content. MDA is an indicator of free radical production and potential to withstand and recover after membrane injury under stress. In our experiment, we established that 40 mM NaCl salinity induced a reduction of the MDA content with 17%, while 80 mM NaCl salinity lead to a decrease of the MDA content with 55%. When measuring the thiol groups, an increase of about 108% of their content was observed at concentration 40 mM NaCl. A much higher free thiol groups' content increase of 151% was established at concentration 80 mM NaCl. The enlargement of the content of free thiol groups is a marker for the presence of injures caused by salinity stress. The H2O2 levels in our experiment indicate that under the conditions of salinity stress the H2O2 content becomes larger; it became 126% at the high salt concentration of 80 mM NaCl. After salinity treatment of soybean plants the ratio of oxidized to total glutathione GSSG/TG increased with approximately 13% at the lower NaCl concentration and of the order of 26 % at the higher concentration. This brings to decreasing of the nitrogen fixing capacity and the plant sustainability.


Table 8. Values of the biochemical parameters of nitrogen fixing soybean plants.

The decrease of the leaf chlorophyll content under salinity stress is a main phenomenon of the plant sensitivity. In our experiments, both the content of chlorophyll a and chlorophyll b decreased at the two salinity levels, the decrease under 80 mM NaCl being larger.

Concluding, it was found that the results from the implementation of the two methods, leaf spectral reflectance and biochemical analysis, revealed that both the NaCl concentrations

λ1 <0.001 14 from 50 <0.001 5 from 50 λ2 <0.001 13 from 50 <0.001 5 from 50 λ3 <0.001 13 from 50 <0.001 6 from 50 λ4 0.0027 15 from 50 <0.001 7 from 50 λ5 <0.001 12 from 50 <0.001 7 from 50 λ6 0.0298 21 from 50 <0.001 8 from 50 λ7 0.3117 22 from 50 <0.001 10 from 50 λ8 0.3048 24 from 50 0.001 15 from 50 Table 7. Significance p-level of the linear DA in the case of 40 mM NaCl and 80 mM NaCl

species exhibit a remarkable increase in the proline content. MDA is an indicator of free radical production and potential to withstand and recover after membrane injury under stress. In our experiment, we established that 40 mM NaCl salinity induced a reduction of the MDA content with 17%, while 80 mM NaCl salinity lead to a decrease of the MDA content with 55%. When measuring the thiol groups, an increase of about 108% of their content was observed at concentration 40 mM NaCl. A much higher free thiol groups' content increase of 151% was established at concentration 80 mM NaCl. The enlargement of the content of free thiol groups is a marker for the presence of injures caused by salinity stress. The H2O2 levels in our experiment indicate that under the conditions of salinity stress the H2O2 content becomes larger; it became 126% at the high salt concentration of 80 mM NaCl. After salinity treatment of soybean plants the ratio of oxidized to total glutathione GSSG/TG increased with approximately 13% at the lower NaCl concentration and of the order of 26 % at the higher concentration. This brings to decreasing of the nitrogen fixing

Stress markers, pigments Control 40 mM NaCl 80 mM NaCl Phenols, μmol/gDW 131.3 ±2.1 106.7 ±1.1 81% (19↓) 54.6±0.9 41.6% (59↓) Proline, nmol/gDW 21.9±0.7 23.7±2.3 108% (8↑) 37.9±1.9 173% (73↑) MDA, nmol/gDW 141.3±5.3 117.5±6.8 83.1% (17%↓) 62.7±5.6 44.3% (55↓) H2O2 , μmol/gDW 2.8±0.06 3.41±0.04 121% (21↑) 6.35±0.02 226% (126↑) Thiol Groups, μmol/gDW 1.03±0.02 2.15±0.07 (108% ↑) 2.59±0.08 (151%↑) Chlorophyll a, mg/g FW 1.22±0.03 1.19±0.02 98%(2↓) 0.94±0.03 79% (21↓) Chlorophyll b, mg/g FW 0.54±0.01 0.38±0.03 70.3%(30↓) 0.34±0.01 63% (37↓) GSSG/TG, % 57.2 64.6 113%(13↑) 21.3 126% (26↑)

Table 8. Values of the biochemical parameters of nitrogen fixing soybean plants.

decreased at the two salinity levels, the decrease under 80 mM NaCl being larger.

The decrease of the leaf chlorophyll content under salinity stress is a main phenomenon of the plant sensitivity. In our experiments, both the content of chlorophyll a and chlorophyll b

Concluding, it was found that the results from the implementation of the two methods, leaf spectral reflectance and biochemical analysis, revealed that both the NaCl concentrations

NaCl 80 mM NaCl

Number of incorrectly classified objects

*pDA*

NaCl 40 mM NaCl 80 mM

Number of incorrectly classified objects

40 mM

*pDA*

salinity of nitrogen fixing soybean plants.

capacity and the plant sustainability.

One dimensional spaces

bring to salinity stress in the nitrogen fixing soybean plants and to decline of the biological nitrogen fixation. The red edge shift to longer wavelengths is an indicator of stress and is correlated with the decreased chlorophyll content and salinity rate.

The course of the averaged fluorescence spectra over 20 control and 20 salinity treated leaves of nitrogen fixing soybean plants used for the second part of the experiments is shown in Fig. 15 (Iliev et al., 2009b). All spectra were normalized against their second maximum. Changes in the spectra of treated leaves against the controls were significant in the forefront and in the spectral range between first and second maximums (680-740 nm).

Fig. 15. Averaged fluorescence spectra of control and treated with 40 mM and 80 mM NaCl nitrogen fixing soybean plants.

The results of the Student's t-criterion and linear DA are displayed in Table 9 and Table 10. For analysis, five fluorescence values for each spectrum (from all 910) in characteristic wavelengths in the spectral range 600-900 nm were selected: 1 (at the middle of the forefront), 2 (first maximum), 3 (at the middle between first and second maximum), <sup>4</sup> (second maximum) and 5 (at the middle of rear slope), see Fig. 11.


Table 9. Significance p-level of the t-criterion for the set of amplitudes of the fluorescence spectra.

Spectral Remote Sensing of the Responses of Soybean Plants to Environmental Stresses 241

Control 40 mM NaCl 80 mM NaCl

450 550 650 750 850 Wavelength, nm

Fig. 16. Averaged spectral reflectance characteristics of control and treated with 40 mM and

Pairs Control 40 mM NaCl 80 mM NaCl compared mean *pSt* mean *pSt* mean λ1/λ1c 11.93 <0,001 10.92 <0.001 9.28 λ2/λ2c 15.74 <0.001 14.21 <0.001 12.61 λ3/λ3c 16.40 <0.001 14.83 <0.001 13.18 λ4/λ4c 4.08 <0.001 4.83 <0.001 3.65 λ5/λ5c 19.27 <0.001 17.23 <0.001 15.37 λ6/λ6c 35.59 0.029 33.59 0.001 33.60 λ7/λ7c 39.47 0.011 38.06 0.167 38.79 λ8/λ8c 47.91 0.152 48.88 0.007 51.73 Table 11. Significance p-level of the t-criterion in the cases of 40 mm NaCl and 80 mm NaCl

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.

0

80 mM NaCl nitrogen fixing soybean plants.

salinity.

10

20

30

Spectral reflectance, %

40

50

60

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 dimensional spaces defined separately by each of the five wavelengths.


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

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