**4. Effect of water deficit on plant metabolism**

The synthesis and breakdown of metabolites to yield energy is required for the many activities that plants depend upon. But, when plants are exposed to drought stress conditions, physiological and metabolic changes occur. Immediate acclimatisation by the alteration of plant morphology is therefore required for plants to be adapted to the changing environments. Whether plants succeed to acclimatise or not, the subsequent phenotypic modifications observed in water stressed plants would be a function of the metabolic changes. In soybean, like other leguminous plants, decrease in the leaf area, number of individual leaves and the total number of branches per plant is normally observed [16, 24]. However, on the metabolic section, water stressed plants experiences a dramatic decrease in photosynthetic rates as a consequence of the modification in photosynthetic structures and chloroplastidic pigments. Chloroplastidic pigments involve all plant pigments such as chlorophylls and carotenoid pigments embedded in the thylakoid membranes of parenchyma mesophylls [18]. These pigments are primary molecules responsible for making sure that light energy from the sun is captured and converted to chemical energy required for metabolism.

This is the main route in which energy used for synthesis of biological products enters our biosphere. Water stress adversely limits this process by inhibiting the functioning of structure serving as primary support for photosynthetic metabolism. According to Kwon and Woo [24] drought reduce photosynthesis by limiting stomatal operations. In line with this report, the soybean plants subjected to water stress (WT 1 and WT 2) kept their stomata closed to reduce transpiration, hence trying to preserve water. The stomatal micrograph in **Figure 3** illustrates closed stomatal apertures (c, d) prepared from leaves collected during the day. The closure of stomata in turn reduces the concentration of CO2 required in the mesophyll for carboxylation process during the manufacturing of photosynthates. This phenomenon was also reported by Dekov et al. [31], Evert and Eichhorn [18], Lopez-Carbonell et al. [30] and Taiz et al. [17]. Additionally, there were significant variations in stomatal density exhibited by the different genotypes.

Water stressed soybean cultivar TGx 1740-2F and LS 677 exhibited low density of stomata with an average of 154 and 106 in WT 1 and WT 2 respectively, among all the cultivars used (**Table 1**). Furthermore, the two TGx cultivars (TGx 1740-2F and TGx 1835-10E) did not show extensive variations in the stomata among all water stressed plants, including the control. The mean leaf areas of the water stressed plants were also significantly lower compared to the control. The decrease in the leaf area of the plants posed negative effects on the rate of photosynthesis by reducing the leaf surface area in which light is captured. Anatomically, water stress also had an effect on leaf mesophyll thickness which also had an impact on photosynthesis. Cramer and Browman [32] attributed this to the changes in the rate of cellular expansion, which was observed in the maize mesophyll tissues when cell division and differentiation appeared affected by drought stress. However, plants growing in soil grounds of very lower water potential possess poor cell formation and expansion. Schuppler et al. [6] also reported this when assessing the effects of water stress on rate of cell division or mitotic activity on wheat leaf tissues. The report indicated that generally, leaf tissue expansion rate is reduced to more than 50% when plants are subjected to drought stress. In terms of physiological response to water stress, the reduction in chlorophyll content index (CCI) in water stressed plants was

recorded, and the decreases in chlorophyll contents varied according to imposed water stress treatments (**Figure 4**). Plant irrigated once in 15 days (WT 2) showed remarkable decrease in

**Figure 3.** Dermal tissue of the leaf of a typical soybean plant. (a) Soybean plant at R4 stage. (b) Field of epidermal cells of a soybean plant. (c) Light micrograph of slightly higher magnification of stomatal complexes on a soybean leaf. (d) Light

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Control plants did not exhibit significant reduction in CCI nor variation in all cultivars' CCI measurements even before when water treatments were imposed on water stressed plants (**Figure 4a**, **b**). But then, differences were not expected in the CCI estimates of control plants measured early during growth and later before termination of the experiment, since the plants were adequately watered. Therefore, as expected the chlorophyll degradation was not induced on control plants as a result of water stress. As the differences in the chlorophyll content and degradation were observed in water stressed plants, these findings were in line with Dhanda et al. [33] and Benjamin and Nielsen [34]'s reports on the effects of drought on plant metabolism. As indicated on Section 2, to examine and confirm the degradation of chlorophyll and its subsequent effects on photosynthetic activity, starch analysis was performed. Leaves detached from randomly selected soybean plants

CCI (**Figure 4d**) than WT 1 plants (**Figure 4b**).

micrograph in the epidermis showing epidermal hairs.

Water Stress: Morphological and Anatomical Changes in Soybean (*Glycine max* L.) Plants http://dx.doi.org/10.5772/intechopen.72899 19

**4. Effect of water deficit on plant metabolism**

18 Plant, Abiotic Stress and Responses to Climate Change

in turn reduces the concentration of CO2

The synthesis and breakdown of metabolites to yield energy is required for the many activities that plants depend upon. But, when plants are exposed to drought stress conditions, physiological and metabolic changes occur. Immediate acclimatisation by the alteration of plant morphology is therefore required for plants to be adapted to the changing environments. Whether plants succeed to acclimatise or not, the subsequent phenotypic modifications observed in water stressed plants would be a function of the metabolic changes. In soybean, like other leguminous plants, decrease in the leaf area, number of individual leaves and the total number of branches per plant is normally observed [16, 24]. However, on the metabolic section, water stressed plants experiences a dramatic decrease in photosynthetic rates as a consequence of the modification in photosynthetic structures and chloroplastidic pigments. Chloroplastidic pigments involve all plant pigments such as chlorophylls and carotenoid pigments embedded in the thylakoid membranes of parenchyma mesophylls [18]. These pigments are primary molecules responsible for making sure that light energy from the

This is the main route in which energy used for synthesis of biological products enters our biosphere. Water stress adversely limits this process by inhibiting the functioning of structure serving as primary support for photosynthetic metabolism. According to Kwon and Woo [24] drought reduce photosynthesis by limiting stomatal operations. In line with this report, the soybean plants subjected to water stress (WT 1 and WT 2) kept their stomata closed to reduce transpiration, hence trying to preserve water. The stomatal micrograph in **Figure 3** illustrates closed stomatal apertures (c, d) prepared from leaves collected during the day. The closure of stomata

during the manufacturing of photosynthates. This phenomenon was also reported by Dekov et al. [31], Evert and Eichhorn [18], Lopez-Carbonell et al. [30] and Taiz et al. [17]. Additionally, there were significant variations in stomatal density exhibited by the different genotypes.

Water stressed soybean cultivar TGx 1740-2F and LS 677 exhibited low density of stomata with an average of 154 and 106 in WT 1 and WT 2 respectively, among all the cultivars used (**Table 1**). Furthermore, the two TGx cultivars (TGx 1740-2F and TGx 1835-10E) did not show extensive variations in the stomata among all water stressed plants, including the control. The mean leaf areas of the water stressed plants were also significantly lower compared to the control. The decrease in the leaf area of the plants posed negative effects on the rate of photosynthesis by reducing the leaf surface area in which light is captured. Anatomically, water stress also had an effect on leaf mesophyll thickness which also had an impact on photosynthesis. Cramer and Browman [32] attributed this to the changes in the rate of cellular expansion, which was observed in the maize mesophyll tissues when cell division and differentiation appeared affected by drought stress. However, plants growing in soil grounds of very lower water potential possess poor cell formation and expansion. Schuppler et al. [6] also reported this when assessing the effects of water stress on rate of cell division or mitotic activity on wheat leaf tissues. The report indicated that generally, leaf tissue expansion rate is reduced to more than 50% when plants are subjected to drought stress. In terms of physiological response to water stress, the reduction in chlorophyll content index (CCI) in water stressed plants was

required in the mesophyll for carboxylation process

sun is captured and converted to chemical energy required for metabolism.

**Figure 3.** Dermal tissue of the leaf of a typical soybean plant. (a) Soybean plant at R4 stage. (b) Field of epidermal cells of a soybean plant. (c) Light micrograph of slightly higher magnification of stomatal complexes on a soybean leaf. (d) Light micrograph in the epidermis showing epidermal hairs.

recorded, and the decreases in chlorophyll contents varied according to imposed water stress treatments (**Figure 4**). Plant irrigated once in 15 days (WT 2) showed remarkable decrease in CCI (**Figure 4d**) than WT 1 plants (**Figure 4b**).

Control plants did not exhibit significant reduction in CCI nor variation in all cultivars' CCI measurements even before when water treatments were imposed on water stressed plants (**Figure 4a**, **b**). But then, differences were not expected in the CCI estimates of control plants measured early during growth and later before termination of the experiment, since the plants were adequately watered. Therefore, as expected the chlorophyll degradation was not induced on control plants as a result of water stress. As the differences in the chlorophyll content and degradation were observed in water stressed plants, these findings were in line with Dhanda et al. [33] and Benjamin and Nielsen [34]'s reports on the effects of drought on plant metabolism. As indicated on Section 2, to examine and confirm the degradation of chlorophyll and its subsequent effects on photosynthetic activity, starch analysis was performed. Leaves detached from randomly selected soybean plants were obtained and taken to the laboratory for starch analysis. The leaves were bleached in boiling 90% ethanol and incubated in dilute iodine (0.5 M) solution (2:1) for 3 minutes and then rinsed with distilled water. Rinsing is necessary to remove excess iodine solution on the leaves while a colour change occurs. The iodine stained leaves (**Figure 5a**–**c**) were then visualised under a ZIESS Discovery V12 stereo microscope mounted with an ICc5 Axio-Camera. The presence of high starch content was observed in the control (**Figure 5c**); whereby starch contents in WT 1 (**Figure 5a**) and WT 2 (**Figure 5b**) were very drastically reduced because of poor photosynthetic activity. Intense blue-black colour on the leaves of control plants indicate the presence of starch, generated from the photosynthesised carbohydrates. Only minor traces of starch were observed from WT 1 and WT 2 leaves due to

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The formation of cell protuberance containing nitrogen-fixing Gram-negative bacteria in the roots of legumes plays an important role in improving plant growth characteristics, crop productivity and maintaining soil fertility. This establishment of lumps on roots of plants

of proteins, nucleic acids and other necessary nitrogen-containing compounds required for plant, animal and human growth and development. However, various reports have indicated that, water stress induces low frequencies of nodulation in many legumes, including soybean. Miao et al. [35] provided evidence that verifies sensitivity of soybean nodulating root cells and Rhizobium to water stress. In 2003, Ramos et al. [36] also indicated that, water stress affect nodulation in other legume species like *Phaseolus vulgaris* L. Failure for soybean roots to produce effective nodulations affect the metabolism of nitrogenous and carbonic compounds in the plant. The changes resulting into decreased nodulation could cause reduction in various aspects of plant growth (stem height, stem wood diameter and root dry weight) due to drought as reported by Shetta [37]. Additionally, Shetta indicated that the initiated nodules can become thickened and more resistant to infection by *Rhizobium* as a result of this stress. Poor nodulation can be induced by poor plant nutrition, seed filling, or abiotic stress factors. In WT 2 plants, where irrigation was withheld for 15 days, nodulation was severely affected (**Figure 6f**). It was found that nodules stopped fixing nitrogen and then started decomposing. Nodulation and nitrogen fixation in the WT 1 also decreased following imposed water deficit stress. The nodules turned green (**Figure 6e**) and this predominant green colour indicated inefficient fixation by Rhizobium strain in contrast to highly efficient red-pinkish nodules in the control (**Figure 6d**). This inefficiency may have been caused by the poor amounts of assimilates that are exchanged from soybeans to the bacteria due to reduced rates of photosynthesis in the leaves. Plants do not get fixed nitrogen from Rhizobium for free. For plants to receive fixed atmospheric nitrogen, in a form that is directly available for growth (nitrates–

was reported by Dupont et al. [38], Serraj et al. [39] and Stajkovic et al. [40] as the major

The symbiosis establishment is playing a very critical role in ecological and agronomic supply

, estimated to account for a total of about 65% of the nitrogen fixed in legumes used for

), plants must give bacteria sugars. This symbiotic relationship

for use in the synthesis

by stabilising C–N ratio.

(known as nodulation) guarantees the supply of fixed atmospheric N<sup>2</sup>

water stress.

NO3

of N2

agriculture globally.

¯ and ammonium–NH4

+

stimulant of increased plant biomass, stabilising atmospheric CO2

**5. Nodulation**

**Figure 4.** Effect of water deficit stress on photosynthetic pigment (chlorophyll) content of soybean plants expressed to CCI. (a) Chlorophyll content of control plants during early growth stages (V3). (b) Leaf chlorophyll content of the control during early reproductive stages. (c) Amount of chlorophyll content in WT 1 plants. (d) Leaf chlorophyll content in WT 2. Data represent CCI means and the different letters denote significant differences of the means at p < 0.05.

**Figure 5.** Iodine test on ethanol bleached leaves. After bleaching and staining with iodine: (a) Show traces of starch on leaflet taken from water stress plants (WT 1). (b) Absence of or minor starch traces on severely water stressed leaflet (WT 2). (c) Starch content (blue black colour) on leaflet taken from the control plants.

on the leaves while a colour change occurs. The iodine stained leaves (**Figure 5a**–**c**) were then visualised under a ZIESS Discovery V12 stereo microscope mounted with an ICc5 Axio-Camera. The presence of high starch content was observed in the control (**Figure 5c**); whereby starch contents in WT 1 (**Figure 5a**) and WT 2 (**Figure 5b**) were very drastically reduced because of poor photosynthetic activity. Intense blue-black colour on the leaves of control plants indicate the presence of starch, generated from the photosynthesised carbohydrates. Only minor traces of starch were observed from WT 1 and WT 2 leaves due to water stress.
