**3.2 Induction of stresses**

The first part of experiments was carried out to investigate the influence of a single environmental stress factor (salinity) on the spectral properties and physiological state of soybean plants. Plants were grown in nutrient solution with constant nitrate levels at all development stages (1st to 4th trifoliate expanded leaves). They were divided into three groups. These one kept only in nutrient solution were used as control (untreated). At the growth stage of 2nd trifoliate expanded leave to the nutrient solutions of the other two groups was added NaCl at concentrations 40 mM and 80 mM. Some of the investigated leaves are shown in Fig. 3. They are from plants salinity treated for 14 days.

Fig. 3. Leaves from control (a) and treated with 40 mM NaCl (b) and 80 mM NaCl (c) soybean plants.

instrument, developed by Raimondi et al. (2007), was successfully employed in summer 2007 during the joint CarboEurope, FLEX, and Sentinel-2 ESA mission campaign. These groundbased active fluorescence-sensing techniques can be used whenever temporal monitoring of

Lately, chlorophyll fluorescence techniques proved to be a non-intrusive, fast and reliable attractive tool in ecophysiological studies, and have extensively been used in assessing plant

The experiments were carried out on young soybean plants (*Glycine max L., cultivar Pavlikeni 101)*. They were grown as water cultures in a growth chamber under controlled conditions (16/8 photoperiod day to night, photon flux density 90 mol m-2 sec-1, humidity 60-70%, and temperature 25 1 C). Soybean seeds were surface sterilized with 70% ethanol and washed afterwards several times in distilled water. Then, they were let to germinate on a damp filter paper at 24 °С for three days in dark. On the 4th day seeds were transferred in plastic vessels with half strength well aerated Helrigel nutrient solution (Helrigel, 1898) and were put to

The first part of experiments was carried out to investigate the influence of a single environmental stress factor (salinity) on the spectral properties and physiological state of soybean plants. Plants were grown in nutrient solution with constant nitrate levels at all development stages (1st to 4th trifoliate expanded leaves). They were divided into three groups. These one kept only in nutrient solution were used as control (untreated). At the growth stage of 2nd trifoliate expanded leave to the nutrient solutions of the other two groups was added NaCl at concentrations 40 mM and 80 mM. Some of the investigated

leaves are shown in Fig. 3. They are from plants salinity treated for 14 days.

Fig. 3. Leaves from control (a) and treated with 40 mM NaCl (b) and 80 mM NaCl (c)

a b c

fluorescence transients is required regardless of the appearance of cloud cover.

responses to environmental stress.

**3. Materials and treatments** 

**3.1 Plant material** 

growth clamber.

soybean plants.

**3.2 Induction of stresses** 

The second part of the experiments was focused on studying of the effect of the salinity on nitrogen fixing soybean plants. Three day's seedlings were inoculated with adding of 108 cells ml-1 suspension of Bradyrhizobium japonicum strain 273. After that they were transferred into plastic vessels with Helrigel nutrient solution. The nitrogen in the nutrient solution was equal to ¼ of the full dose until the growth stage of fully expanded 2nd trifoliate leaf, i.e. up to nodule forming and beginning of effective nitrogen fixation. The plants were salinity treated during the vegetative stage of growth from 2nd to 4th trifoliate expanded leaf for 14 days. Salinity was performed by means of adding in nutrient solution NaCl at two concentrations (40 mM and 80 mM). The control plants were kept only in nutrient solution. Figs. 4, 5, and 6 show the roots and nodules of nitrogen fixing control and treated with two NaCl concentrations plants on 14th day after the salinity.

The third part of experiments was aimed to investigate the effect of salinity stress and two consecutive stress factors (salinity and UV-B radiation) on young nitrogen fixing soybean plants. They were grown, nitrogen fixing, and salinity treated in the same way as the plants of the previous experiments.

Fig. 4. Control nitrogen fixing soybean plants: a) leaves; b) roots.

The investigated plants were divided into six groups. The first group consisted of untreated (control) plants. The second and third groups were only salinity treated by two NaCl concentrations. The plants of the fourth group were control (not salinized). The plants of fifth and sixth groups were treated for 14 days with 40 mM and 80 mM NaCl, respectively. Together with the control group they were irradiated with UV-B light for two hours. As a light source a lamp HPQ type with intensity 64.4 μmol m-2 s-1 was used at a distance of 25 cm. Fig. 7 a), b) shows some of investigated leaves from first (control for salinity treatment) and forth (control for treatment with UV-B radiation) groups. Fig. 8 a), b) and Fig. 9 a), b) show some leaves from plants treated with 40 mM NaCl and (40 mM NaCl + UV-B), and 80 mM NaCl and (80 mM NaCl + UV-B), respectively.

Spectral Remote Sensing of the Responses of Soybean Plants to Environmental Stresses 227

 Fig. 7. Leaves from plant groups: a) first (control); b) fourth (control +UV-B).

a) b)

Fig. 9. Leaves from plant groups: a) third (80 mM NaCl salinity; b) sixth (80 mM NaCl + UV-B)

UV-B).

a)

a)

Fig. 8. Leaves from plant groups: a) second (40 mM NaCl salinity; b) fifth (40 mM NaCl +

b)

b)

Fig. 5. Nitrogen fixing soybean plants treated with 40 mM NaCl: a) leaves; b) roots.

Fig. 6. Nitrogen fixing soybean plants treated with 80 mM NaCl: a) leaves; b) roots.

Fig. 5. Nitrogen fixing soybean plants treated with 40 mM NaCl: a) leaves; b) roots.

a b

a b

Fig. 6. Nitrogen fixing soybean plants treated with 80 mM NaCl: a) leaves; b) roots.

Fig. 7. Leaves from plant groups: a) first (control); b) fourth (control +UV-B).

Fig. 8. Leaves from plant groups: a) second (40 mM NaCl salinity; b) fifth (40 mM NaCl + UV-B).

Fig. 9. Leaves from plant groups: a) third (80 mM NaCl salinity; b) sixth (80 mM NaCl + UV-B)

Spectral Remote Sensing of the Responses of Soybean Plants to Environmental Stresses 229

Pigment contents (chlorophyll a and b) were calculated after the extraction of leaf material with 80% acetone according to the method of Arnon (1949). The extinction of chlorophyll a and chlorophyll b was determined at 663 nm and 845 nm using a spectrometer Specol 11.

Hyperspectral reflectance data were collected in VIS and NIR spectral ranges (450-850 nm) by using a portable fibre-optics spectrometer USB2000 (http://www.OceanOptics.com). In the range investigated the main part of the reflected from leaves radiation is concentrated. Data were obtained at 1170 spectral bands with a step of 0.3 nm and a spectral resolution of

The reflectance measurements were carried out on an experimental setup in laboratory (Fig. 10). The entrance lens, a standard screen WS1 (diffuse reflectance white plastic with Lambertian reference surface) and plant leaves were set on a special adjustable platform, which provides an accurate relative positioning of all equipment components. The fibreoptic cable was located at nadir view (perpendicular to leaf surface). As a light source a halogen lamp providing homogeneous illumination of measured leaf surfaces was used. The acquisition and processing of data were carried out by means of portable computer using specialized software. The spectral reflectance characteristics were obtained as a ratio of the reflected radiation from the leaves and the reflected radiation from WS1 screen. The measurements were performed on fresh, immediately picked off soybean leaves at the stage

of 4th trifoliate expanded leaf from up to 25 plants from each investigated group.

Fig. 10. Experimental setup for spectral reflectance measurements.

**5. Spectral measurements 5.1 Leaf spectral reflectance** 

1.5 nm.
