**7. Other biotic and abiotic stress effects**

Plants are normally adapted to grow in complex and diverse environments. The success in growth establishment, reproduction and productivity of plant species rely upon a set of environmental conditions, natural resources and the interactions (beneficial or harmful) that exist among plants and other organisms. However, certain types of interactions, especially those including biological factors such as insects, parasites, viruses and bacterial pathogens have detrimental effects on plants. In addition, the non-living physical or chemical factors such as light, temperature, salinity, water, nutrient and other variables that can be found in the aquatic or terrestrial ecosystem also have major impacts on plant life. All above-mentioned factors may induce plant stress, defined by Taiz et al. [17] as a condition that prevent a given plant from achieving its maximum growth and reproductive potential as measured by vegetative growth, flowering, seed formation and yield quantity. Gerhardson [45] gave further information by providing more insights on disease symptoms caused by pathogenic strains of *Fusarium*, *Cylindrocarpon*, *Phoma* and *Pythium* mostly on legume crops. Strains of the genera *Pythium* have also been found to cause seedling mortality in cowpea [46]. These soil-borne legume pathogens, including other wide spread disease causing fungi; induce root, stem and leaf rots in pea, beans and alfalfa [45]. Abiotic environmental stress dramatically affects growth and productivity of many cereals, oilseeds, vegetables and fruit crops.

many regions worldwide have led to tremendous adversities on agriculture, biodiversity, wildlife and subsequently, the well-beings of many people. Plants normally evolve in order to adapt and adjust to the low water conditions or any other biotic and abiotic constraint. These adaptive measures are an important event of evolution in the history of life, with far reaching consequences as described by Kenrick and Crane [55]. However, this is a very slow process in nature, even if it may result in greater diversity of plants, making changes in plants at their physiological, biochemical and molecular levels. These changes show a wide range of adaptations, at different levels in which plants attempt to deal with drought stress [56]. Plants manage water stress in various ways. They regulate stomatal closure to reduce water loss, especially through transpiration. The stomatal opening and closing is very essential for gaseous exchange as reported by Osakabe et al. [57]. They are controlled by complex regulatory events mediated by abscisic acid (ABA) signalling and ion transport induced by abiotic stress. Nonetheless, stomata closure negatively affects the rates of photosynthetic metabolism by lowering the

. Plants also alter metabolic functions in order to inhibit the production of reac-

¯), and H2

photorespiration from the Calvin cycle. However, some succulent plants use Crassulaceae acid

dependent process) and in the specialised cell around the leaf veins called the bundle sheath

produce oxaloacetate which is converted into malate, transported into the bundle sheath for

water use. In addition to all of the metabolic strategies mentioned above, modern genetic engineering technology can be used. This technology is focused on breeding biotic/ abiotic stress tolerant plants. The biotechnological approaches such as *Agrobacterium*-mediated genetic transformation allow manipulation of the host plant's genome for the expression of foreign genes required in the plant stress response. This technique was initially used to isolate genes used for stress tolerance in *Arabidopsis*. This plant was only used as a model plant and has played an important role in elucidating the basic processes constituting the expression of regulatory genes for stress tolerance [61]. The insights from research on *Arabidopsis* have been used in attempts of unravelling biotic/ abiotic stress effects in plants, subsequently resulting in the development of transgenic plants tolerant to drought, salinity and chilling stress. Montero-Tavera et al. [62] reported upregulation of a number of genes in two common bean varieties with different susceptibility to drought stress. Variety Pinto Villa was relatively susceptible than cultivar Carioca. The reports indicated that drought tolerant variety displayed a more developed root vascular tissue system under stress conditions, when compared to the other non-transgenic cultivars. Differential root phenotype showing variations in root lengths, surface area and fineness of the root system was also reported by Abenavoli et al. [63]. In soybean, stress tolerant genes were introduced and DREB or ARED genes expressed to show improved tolerance to water stress under greenhouse conditions [11, 64]. The genetic

O2

Water Stress: Morphological and Anatomical Changes in Soybean (*Glycine max* L.) Plants

with minimum loss of water. For example; many C3

is fixed in the mesophyll spongy cells (a light-

and CAM plants are well adapted to hot, dry envi-

plants lack strategies to efficiently and effectively manage

minimising photorespiration thus, saving water.

plants like peanut (*Arachis hypogaea*), potato (*Solanum tuberosum*) and

[52, 58]. Other changes involve

http://dx.doi.org/10.5772/intechopen.72899

molecules by separating

plants and they

plants

25

amount of CO2

tive oxygen species (ROS) such as superoxide (O2

In monocots such as maize and wheat, CO2

use in Calvin cycle [17, 59, 60]. Both C4

soybean (*Glycine max* L.). These C3

ronments than the C3

do not have photosynthetic adaptations to reduce the loss of CO2

(light-independent). These monocotyledonous plants are referred to as C4

development of strategies to fix CO2

metabolism (CAM) to salvage CO2

Oilseeds such as soybeans have suffered major losses from the short and prolonged occurrence of abiotic stress, especially drought, extreme temperatures, flooding and waterlogging [47]. Plants experiencing drought stress may also endure other stress effects simultaneously, like salinity and heat stress. Multiple stress effects and symptoms may be concomitantly induced by the occurrence of a single stress as described by Miransari [48] leading to combinational abiotic stress. In soybean, drought stress has many negative consequences ranging from reduced production of signalling and communication metabolites, decreased photosynthetic assimilates, nutrient deficiency, accumulation of reactive oxygen species (ROS) and reduction in nitrogen (N) fixation by affecting symbiotic relationship with *Rhizobium* species [49–52]. Soybean is an important crop for the production of oils and proteins used for feed and human consumption. It is a potential source for biodiesel and has been used to manufacture a number of pharmaceutical products [53]. But, the high sensitivity to water deficit stress shown in this study by this crop encourage the development of stress tolerant soybean varieties. Drought and other growth constraints are inevitable consequences of climate change. Therefore, investigation on the physiological, anatomical and morphological response of soybean to these biotic and abiotic constraints is highly recommended.

#### **8. Water stress management and crop improvement**

As previously discussed, drought stress is the most widely known and devastating stress factor that limit plant growth, development and productivity. Khaine and Woo [54] reported that, frequent drought effects recently and currently experienced, are largely induced by the changes in climatic conditions. The continuously fluctuating meteorological conditions in many regions worldwide have led to tremendous adversities on agriculture, biodiversity, wildlife and subsequently, the well-beings of many people. Plants normally evolve in order to adapt and adjust to the low water conditions or any other biotic and abiotic constraint. These adaptive measures are an important event of evolution in the history of life, with far reaching consequences as described by Kenrick and Crane [55]. However, this is a very slow process in nature, even if it may result in greater diversity of plants, making changes in plants at their physiological, biochemical and molecular levels. These changes show a wide range of adaptations, at different levels in which plants attempt to deal with drought stress [56]. Plants manage water stress in various ways. They regulate stomatal closure to reduce water loss, especially through transpiration. The stomatal opening and closing is very essential for gaseous exchange as reported by Osakabe et al. [57]. They are controlled by complex regulatory events mediated by abscisic acid (ABA) signalling and ion transport induced by abiotic stress. Nonetheless, stomata closure negatively affects the rates of photosynthetic metabolism by lowering the amount of CO2 . Plants also alter metabolic functions in order to inhibit the production of reactive oxygen species (ROS) such as superoxide (O2 ¯), and H2 O2 [52, 58]. Other changes involve development of strategies to fix CO2 with minimum loss of water. For example; many C3 plants do not have photosynthetic adaptations to reduce the loss of CO2 molecules by separating photorespiration from the Calvin cycle. However, some succulent plants use Crassulaceae acid metabolism (CAM) to salvage CO2 minimising photorespiration thus, saving water.

**7. Other biotic and abiotic stress effects**

24 Plant, Abiotic Stress and Responses to Climate Change

Plants are normally adapted to grow in complex and diverse environments. The success in growth establishment, reproduction and productivity of plant species rely upon a set of environmental conditions, natural resources and the interactions (beneficial or harmful) that exist among plants and other organisms. However, certain types of interactions, especially those including biological factors such as insects, parasites, viruses and bacterial pathogens have detrimental effects on plants. In addition, the non-living physical or chemical factors such as light, temperature, salinity, water, nutrient and other variables that can be found in the aquatic or terrestrial ecosystem also have major impacts on plant life. All above-mentioned factors may induce plant stress, defined by Taiz et al. [17] as a condition that prevent a given plant from achieving its maximum growth and reproductive potential as measured by vegetative growth, flowering, seed formation and yield quantity. Gerhardson [45] gave further information by providing more insights on disease symptoms caused by pathogenic strains of *Fusarium*, *Cylindrocarpon*, *Phoma* and *Pythium* mostly on legume crops. Strains of the genera *Pythium* have also been found to cause seedling mortality in cowpea [46]. These soil-borne legume pathogens, including other wide spread disease causing fungi; induce root, stem and leaf rots in pea, beans and alfalfa [45]. Abiotic environmental stress dramatically affects

growth and productivity of many cereals, oilseeds, vegetables and fruit crops.

soybean to these biotic and abiotic constraints is highly recommended.

**8. Water stress management and crop improvement**

Oilseeds such as soybeans have suffered major losses from the short and prolonged occurrence of abiotic stress, especially drought, extreme temperatures, flooding and waterlogging [47]. Plants experiencing drought stress may also endure other stress effects simultaneously, like salinity and heat stress. Multiple stress effects and symptoms may be concomitantly induced by the occurrence of a single stress as described by Miransari [48] leading to combinational abiotic stress. In soybean, drought stress has many negative consequences ranging from reduced production of signalling and communication metabolites, decreased photosynthetic assimilates, nutrient deficiency, accumulation of reactive oxygen species (ROS) and reduction in nitrogen (N) fixation by affecting symbiotic relationship with *Rhizobium* species [49–52]. Soybean is an important crop for the production of oils and proteins used for feed and human consumption. It is a potential source for biodiesel and has been used to manufacture a number of pharmaceutical products [53]. But, the high sensitivity to water deficit stress shown in this study by this crop encourage the development of stress tolerant soybean varieties. Drought and other growth constraints are inevitable consequences of climate change. Therefore, investigation on the physiological, anatomical and morphological response of

As previously discussed, drought stress is the most widely known and devastating stress factor that limit plant growth, development and productivity. Khaine and Woo [54] reported that, frequent drought effects recently and currently experienced, are largely induced by the changes in climatic conditions. The continuously fluctuating meteorological conditions in In monocots such as maize and wheat, CO2 is fixed in the mesophyll spongy cells (a lightdependent process) and in the specialised cell around the leaf veins called the bundle sheath (light-independent). These monocotyledonous plants are referred to as C4 plants and they produce oxaloacetate which is converted into malate, transported into the bundle sheath for use in Calvin cycle [17, 59, 60]. Both C4 and CAM plants are well adapted to hot, dry environments than the C3 plants like peanut (*Arachis hypogaea*), potato (*Solanum tuberosum*) and soybean (*Glycine max* L.). These C3 plants lack strategies to efficiently and effectively manage water use. In addition to all of the metabolic strategies mentioned above, modern genetic engineering technology can be used. This technology is focused on breeding biotic/ abiotic stress tolerant plants. The biotechnological approaches such as *Agrobacterium*-mediated genetic transformation allow manipulation of the host plant's genome for the expression of foreign genes required in the plant stress response. This technique was initially used to isolate genes used for stress tolerance in *Arabidopsis*. This plant was only used as a model plant and has played an important role in elucidating the basic processes constituting the expression of regulatory genes for stress tolerance [61]. The insights from research on *Arabidopsis* have been used in attempts of unravelling biotic/ abiotic stress effects in plants, subsequently resulting in the development of transgenic plants tolerant to drought, salinity and chilling stress. Montero-Tavera et al. [62] reported upregulation of a number of genes in two common bean varieties with different susceptibility to drought stress. Variety Pinto Villa was relatively susceptible than cultivar Carioca. The reports indicated that drought tolerant variety displayed a more developed root vascular tissue system under stress conditions, when compared to the other non-transgenic cultivars. Differential root phenotype showing variations in root lengths, surface area and fineness of the root system was also reported by Abenavoli et al. [63]. In soybean, stress tolerant genes were introduced and DREB or ARED genes expressed to show improved tolerance to water stress under greenhouse conditions [11, 64]. The genetic transformation of many crops, including soybean via *in vitro* or *in vivo* transformation techniques is still very difficult to achieve, despite the aforementioned successes. Several drought tolerant cultivars have been reported in rice (*Oryza sativa*), maize (*Zea mays* L.) and kidney bean (*Phaseolus vulgaris* L.) by Liu et al. [65], Saijo et al. [66] and Shou et al. [67]. The methods used for genetic transformation of these crops are continuously optimised to establish efficient and reproducible protocols using *Agrobacterium tumefaciens*. Lastly, agronomic practices such as reduction of water loss from irrigation systems, minimising water inputs and increasing crop water use efficiency can also be employed to manage water stress [68].

**References**

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[1] Mattson JW, Haack RA. The role of drought in outbreak of plant eating insects. Bioscience.

Water Stress: Morphological and Anatomical Changes in Soybean (*Glycine max* L.) Plants

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[2] Wrather A, Koenning S. Effects of diseases on soybean yields in the United States 1996

[3] Liu S, Yan D, Wang J, Wang B, Wang G, Yang M. Drought mitigation ability index and application based on balance between water supply and demand. Water. 2015;**7**:1792-1807

[4] Petit JR, Jouzel J, Raynaud D, Barkov NI, Barnola JM, Basile I, Bender M, Chappellaz J, Davis J, Delayque G, Delmolte M, Kotlyakov VM, Legrand M, Lipenkov V, Lorius C, Pepin L, Ritz C, Saltzman E, Stievenard M. Climate and atmospheric history of the past

[5] Shao HB, Chu LY, Jaleel CA, Zhao CX. Water-deficit stress-induced anatomical changes

[6] Schuppler U, He PH, John PCL, Munns R. Effect of water stress on cell division and cell-division-cycle 2-like cell-cycle kinase activity in wheat leaves. Plant Physiology. 1998;**177**:667-678

[7] Jewell MC, Campbell BC, Godwin ID. Transgenic plants for abiotic stress resistance. In: Kole C, Michler C, Abbott AG, Hall TC, editors. Transgenic Crop Plants: Volume 1; Principles and Development. Berlin Heidelberg: Springer-Verlag; 2010. pp. 76-132

[8] Lisar SYS, Motafakkerazad R, Hossain MM, Rahman IMM. Water stress in plants: Causes, effects and responses. In: Rahman MM, Hasegawa H, editors. Water Stress.

[9] Food, Agriculture and Natural Resources (FANR). 2017. Food security; early warning system. Directorate for the Southern African Development Community (SADC). Issue no. 3

[10] Food and Agriculture Organization of the United Nations (FAO). 2015. Monitoring agriculture drought with remote sensing data. FAO Agriculture Stress Index System

[11] Ishaq MN, Ehirim BO. Improving soybean productivity using biotechnology approach

[12] Cornelissen JHC, Lavorel S, Garnier E, Diaz S, Buchmann N, Gurvisch DE, Reich PB, Steege H, Morgan HD, van der Heijden MGA, Pausa JG, Poorter H. A handbook of protocols for standardized and easy measurements of plant functional traits worldwide.

[13] Yeung EC. A Beginner's Guide to the Study of Plant Structure. Calgary Alberta, Canada:

[14] Richter GL, Junior AZ, Streck NA, Guedes JVC, Kraulich B, Da-Rocha TSM, Winck JEM, Cera JC. Estimating leaf area of modern soybean cultivars by a non-destructive method.

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**3. Results and Discussion**

#### *3.1. Pigments*

We propose the use of certain pigments on the basis of qualitative observation of MA-XRF maps, hyperspectral distribution maps and associated characteristic reflectance curves. In the case of Prussian blue and cadmium red the extraction of representative spectra from hypserspectral maps supported the identification of pigments. The earliest known manufacturing dates for potential pigments (Table 1) were used to propose *terminus post quem* for the prints. XRF data can be used to calculate elemental concentrations and therefore quantitatively identify the presence of certain elements; however, in our case approximate identifications were sufficient to address research questions concerning the prints' provenance. Summaries of all proposed identifications are given in Appendix A and data supporting this study are available in the associated supplementary information file.

**Table 1.** Earliest known date of commercial availability for pigments used to date the prints in NU Special Collections.


*2.3. Isolation of Symbiodiniaceae Cells*

From each of the 66 samples preserved in 100% ethanol, 1 cm fragments were cut, amounting to an estimated 20 mg of tissue for both species. Each fragment included about two polyps in the case of *S. maldivensis or* about eight polyps in the case of *C. abies.* Following a method adapted from [45] to isolate Symbiodiniaceae cells from cnidarians, samples were placed in 1.5 mL tubes with 500 μL of a 2 M sodium hydroxide solution. These were incubated at 37 ◦C for 1 h and then vortexed at a medium speed (using an Analitik Jena Tmix homogenizer) at the same temperature for about 2 h until complete lysis of the antipatharian tissues had occurred. The tubes were then centrifuged at 8000 rpm for 3 min, and the supernatant was discarded, then the pellet was resuspended in ultra-pure (Milli-Q) water and vortexed. One drop of lugol was added to the solution to re-stain the cells that might have lost pigments after preservation. About 8 μL of the pellet in 7 μL was used per chamber. Counts of visible Symbiodiniaceae were undertaken using a glass haemocytometer and an Axioscope A1 (Zeiss) light microscope. The number of the microalgae cells in all nine squares (1 mm2 each) of four counting chambers per sample was counted in order to estimate the density of cells per μL. To corroborate the efficiency of the procedure, the isolation protocol was also carried out on fresh and preserved (100% ethanol) samples of the scleractinian coral *Seriatopora hystrix* (Figure 3). To test for differences in microalgae density between antipatharian species, depths and colony region (top, middle or bottom part), a quasi-Poisson generalised linear model (GLM) was fitted using the 'stats' package in R [46]. The power to detect differences between depths was calculated using the function 'power.t.test' in the same 'stats' package.

**Figure 3.** Light microscopy images of Symbiodiniaceae-like extracts obtained through the sodium hydroxide isolation method. (**a**) Extract from the antipatharian *Cupressopathes abies* showing only one Symbiodiniaceae-like cell (arrow) but abundant cnidocytes. (**b**) Extract from the scleractinian *Seriatopora hystrix* showing numerous Symbiodiniaceae-like cells, corroborating the efficiency of the isolation method. Scale bars = 10 μm.

Histological sections were taken to determine the presence and location of Symbiodiniaceae cells within the coral's tissue. About 1.5 cm of each sample was dehydrated using increasing concentrations of ethanol, then soaked in butanol at 60 ◦C for 24 h. These samples were then embedded in liquid paraffin. Serial sections of 7 μm were cut using a Microm HM 340E (Zeiss) microtome and stained with a Masson's trichrome. The sections were observed and photographed using an Axioscope A1 (Zeiss) light microscope and Axiocam 305 camera.