Keywords

5.1 Introduction

Legumes are widely grown for food, forage, feed, fiber, medicinal and industrial compounds, flowers and other uses in the whole world. The great agricultural and environmental role of legumes lies in their potential to establish symbiotic association with rhizobia which makes legume a target crop in sustainable farming. Accordingly, using beneficial soil microorganisms is the established, promising, and sustainable low-input soil management ventures. In nature, majority of plants along with legumes possess mycorrhizal associations. Arbuscular mycorrhizal fungus mainly affects the nodulated legumes, the biodiversity of the neighboring soil, and associated non-leguminous plants that make them most competent ecological factor in the ecosystem functions. Roots of leguminous plants are occupied and established with rhizobia (Spaink 1996) along with mycorrhiza (Harrison 1999). Legumes are also essential for agroforestry, an important area essential for sustainable agriculture. In addition, agroforestry practices have been proved useful in conserving the mycorrhizal inoculum in soils (Cardoso et al. 2003). Therefore, nutrient-acquisition symbiosis between soil microorganisms and plants are important to plant evolution and ecosystem function (Dakora and Phillips 2002; Simms and Taylor 2002).

Successful plant-microbe relations in the rhizospheric zone are most important agent for plant healthiness and fertility of soil (Jeffries et al. 2003). Arbuscular mycorrhizal fungus, which forms symbiosis with majority of plants, influence plant community development, nutrient’s uptake, water relations, and above-ground productivity (Gosling et al. 2006; Van der Heijden et al. 2008). Mycorrhizal legumes also play key role in rehabilitation of ruined lands and deserted habitats affirming the ecological importance of this marvelous relationship (Requena et al. 2001; Valdenegro et al. 2001). Due to these characteristics use of arbuscular mycorrhizal fungus inoculum are proving suitable alternative to agrochemicals. Arbuscular mycorrhizal fungus is also very much helpful against various stresses (biotic and abiotic) as bioprotectants (Van der Heijden et al. 2008). For legumes, arbuscular mycorrhizal fungus has also beneficial effects for ecophysiology, biota of adjoining soil and connected non-leguminous plants (Bethlenfalvay and Newton 1991). Arbuscular mycorrhizal fungus is marvelous and efficient ecological factor for improving growth along with N content of legumes (Azcon-Aguilar et al. 1979). Arbuscular mycorrhizal fungus and rhizobia are important symbiotic associations of natural ecosystems which influence productivity, nutrition and resistance of plants (Demir and Akkopru 2007; Matiru and Dakora 2004). Legumes have symbiotic relations with both Arbuscular mycorrhizal fungus and rhizobia (Lodwig et al. 2003; Sprent 2001). Symbiotic associations of these microorganisms with higher plants plays vital role during weathering of minerals and rocks, ultimately contributing to pedogenesis (Lambers et al. 2009). In the rhizosphere, N2 fixing activity of rhizobia also induced in plants having mycorrhizal association, and synergistic interactions have been noticed in various studies (Barea et al. 2002; Van der Heijden et al. 1998, 2006).

Use of both symbionts together in pot and field experiments resulted in better P and N acquisition along with increased plant biomass, although effects were due to specific composition of symbiont (Requena et al. 2001; Subba Rao et al. 1986; Xavier and Germida 2002). Under poor availability of P and N, arbuscular mycorrhizal fungi (AMF) play key role in transport of nutrients from host plant to adjacent plants through mycorrhizal hyphae (Heap and Newman 1980). Exchange of assimilates between the plants may also occurs through hyphae of AMF and also proved in various studies with the help of 14C (Brownlee et al. 1983; Francis and Read 1984; Reid and Woods 1969). Assimilates transfer through hyphal linkages were also proven during the 32P experiment (Chiariello et al. 1982). More specifically, different experimental results have also verified the allocation of fixed N2 from legume mycorrhizal plants to nearby or adjacent non-leguminous plants via active hyphal connections (Hamel et al. 1991; Li et al. 2009). However, arbuscular mycorrhizal fungus vary in their capability to deliver plant nutrients (Ghosh and Verma 2006; Van der Heijden et al. 2003) signifying more production of appropriate strains towards sustainable agricultural practices.

Various technologies regarding production of plant growth promoting rhizobia and arbuscular mycorrhizal fungus is now available commercially for exploiting the profits of these microorganisms in horticulture, agriculture and in restoration of ruined ecosystems (Barea et al. 2005). Similarly, symbiotic association of legume-mycorrhiza-rhizobia has revealed improvements in leguminous plants, leading to better growth and yield (Babajide et al. 2008; Jia et al. 2004; Wu et al. 2009). Moreover, several investigations have also shown the benifits of arbuscular mycorrhizal fungus during various stresses (abiotic/biotic) in legumes (Aysan and Demir 2009; Khan 2006; Rabie and Almadini 2005; Shokri and Maadi 2009; Vivas et al. 2003).

Several ecological factors now days are proving harmful to the establishment of rhizobial and mycorrhizal symbiosis in plants, out of which UV-B radiation is an important issue. In the past few decades, an enhancement in UV-B coming to Earth’s surface is of serious concern to the climate change globally (Caldwell et al. 1989; Madronich et al. 1995). This increase is noticed due to manmade uses of oxygen, nitrogen, hydrocarbon, fluorine and chlorine compounds, which appeared in the 1930s having the main characteristic of high affinity of reaction with O3 in the stratosphere. This initiated the destruction of stratospheric O3 detected in the 1970s (Rowland 2006; WMO 2007), and its function of filtrating Ultraviolet-B light is weakened. Besides emission of chlorofluorocarbons, halons and methyl chloroforms, Anderson et al. (2012) observed that water vapours in stratosphere is also causing O3 depletion leading to increments in solar UV-B on the Earth’s surface. Besides seven chlorofluorocarbons and six hydrochlorofluorocarbons, Laube et al. (2014) have reported other three chlorofluorocarbons and one hydrochlorofluorocarbon, are damaging stratospheric O3. As a consequence, UV-B (280–315 nm) coming to the biosphere has been increased.

In recent era, the ongoing reduction of O3 layer and consequent increments in UV-B on Earth’s surface is a chief global issue posing serious threat to life due to alteration of diverse range of biological and ecological processes (Caldwell et al. 2003; Kakani et al. 2003a, b; McKenzie et al. 2003). Although the effects might be more dramatic in the Antarctic, there are significant signs of increasing UV-B in the temperate and tropical regions (Caldwell et al. 1995; Rosenfield and Schoeberl 2005; McKenzie et al. 2007). Since, ambient UV-B levels in the tropics are already high, any further enhancement in UV-B due to further decrease in O3 level is of great concern for tropical regions situated in low O3 belt including India, and is predicted to receive high UV-B flux in future also (Sahoo et al. 2005; Tandon and Attri 2011; Anderson et al. 2012).

Many scientific reports regarding UV-B effects were generally focused on aboveground traits of terrestrial plants, while indirect impacts regarding belowground and associated plant performances are lacking. Long term impact of such modifications can cause restructuring of the ecosystem (Caldwell 1997). UV-B, although, doesn’t affect belowground processes directly but its indirect impacts cannot be ignored (Fig. 5.1). Recently, Choudhary et al. (2013) showed the detrimental effects of elevated UV-B in the mung bean and Lou et al. (2011) in barley rhizospheres. Therefore, understanding the effect of environmental factors mimicking the future would be helpful in assessing growth and yield of vital agricultural crops. This review aims to summarize various modifications in plant due to UV-B and to find out the possible reasons responsible for loss in symbiotic associationship (rhizobial and mycorrhizal) (Fig. 5.1). The review also highlights various additional UV-B impacts on above ground and below ground portions of leguminous plants.

Fig. 5.1
figure 1

Schematic presentation demonstrates the depletion of stratospheric ozone layer and penetration of harmful UV-B radiations on the Earth’s surface affecting various aboveground traits of leguminous plants. Leguminous plants in particular encounter the indirect impact of UV-B to belowground functionings. UV-B affecting microbial population of the rhizosphere through altered exudation of phenols and flavonoids and finally affecting nodulation as well as mycorrhizal association. CFCs chloroflurocarbons, PAL phenylalanine ammonia lyase, CHS chalcone synthase

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5.2 Effects of Elevated UV-B on Leguminous Plants

Extensive studies were performed during past three decades on leguminous plants with UV-B using different environmental conditions. We have compiled the summary of the available work regarding UV-B impacts on leguminous plants in Table 5.1. Here, we have provided effects on visual manifestations, anatomy of leaf and its ultrastructure, growth, development, UV-B protecting compounds, photosynthetic pigments, photosynthesis and yield. Plant performance against UV-B is related to balance between cellular damage and its repair and adaptation mechanisms. Therefore, to assess UV-B sensitivity and tolerance of various species and cultivars of plants, growth is considered as a good parameter (Jansen et al. 1998; Frohnmeijer and Staiger 2003). The primary molecular targets are the bases for the damaging and adaptive effects at leaf and plant level. Harmful impacts of the high-energy UV-B radiation might results in photodestruction of biomolecules like DNA and proteins. When molecular processes in the cells are triggered and regulated directly or indirectly by UV-B light then the initiation of repair and adaptation processes like induction of flavonoids and phenolics, curling and cupping of leaves, deposition of wax contents and modifications in trichomes were reported. The balance of these deleterious and UV-B regulated cell processes determines the UV-B effects at plant level as the plant performance (Jansen et al. 1998).

Table 5.1 Effect of ultraviolet-B radiation on morphological, physiological, biochemical and yield characteristics of various leguminous plants under different experimental conditions
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5.2.1 Deleterious Effects of UV-B

High UV-B doses are harmful for cellular components. This high energy radiation is causing photochemical changes to important biomolecules such as proteins and nucleic acids (Jansen et al. 1998, Greenberg et al. 1996). High UVB can also induce the reactive singlet state oxygen molecule (1O), which leads to tissue damage (Greenberg et al. 1996). The photodestruction of biomolecules leads to several direct UV-B effects like DNA damage. This damage may result in general stress responses such as wound signalling or repair mechanisms, reduced plant growth or decreased fecundity which is indirect regulated effects (Brosche and Strid 2003; Frohnmeijer and Staiger 2003, Jansen 2002). DNA is very susceptible molecular target of UV-B with far reaching consequences. The major damage is cyclobutane pyrimidine dimers and 6–4′ photoproducts formation (Jordan 1996). When the damage is not repaired, DNA replication and gene transcription are blocked. This results in mutations, a disturbed cell cycle and disrupted cellular metabolism (Brosche and Strid 2003, Jansen et al. 1998, Jordan 1996). Proteins absorb UV-B and the ubiquitous function of proteins in the cell can affect nearly all cell processes, metabolism and cell structures. For instance Rubisco in Calvin cycle may be inactivated by UV-B leading to reduced photosynthesis (Greenberg et al. 1996). Also photosystem II, thylakoid membrane, chlorophyll and carotenoids can be damaged or inactivated so that photosynthesis is reduced.

Other molecular targets are biomembranes; especially the polyunsaturated fatty acids are vulnerable to oxidative damage. Peroxidation of lipids can take place which causes disturbances in biomembrane function and its permeability. Also ATPases in the membrane can be inhibited by UV-B (Greenberg et al. 1996). Finally, phytohormones, especially indoleacetic acid are targets for UV-B. Indoleacetic acid is important for apical dominance and cell elongation. Therefore, altered indoleacetic acid levels have consequences for plant morphology (Jansen 2002). Concentrations of indoleacetic acid might be reduced directly via photooxidation but also indirectly, by binding of indoleacetic acid to flavonoids. Till now it is elucidated if the indoleacetic acid levels are regulated via UV-B specific receptor and if the levels are result of photochemical reactions. Therefore, plant alterations regulated by indoleacetic acid could be a specific UV-B adaptive response, which occurs after damage to indoleacetic acid (Jansen 2002). How these changes in leguminous plants are harmful for symbiotic relationship has been explained in further sections of this review.

5.2.2 Adaptations in Response to UV-B

As plants lack mobility, and unable to escape UV-B stress. Therefore, they possess several repair and adaptation mechanisms to overcome the harmful impact of UV-B. Adaptation to UV-B generally consists of responses like an altered plant growth, plant architecture and leaf morphology, enhanced flavonoid biosynthesis, enhanced capacity for reactive oxygen species scavenging and DNA repair etc. Plants can adapt to UV-B by attenuating, before this radiation can be harmful to photosynthetic cells and the nucleus. This attenuation is possible at the entire plant level by a more stunted morphology such as shorter plants and internodes with increased branching (Krizek 2004, Day and Neale 2002). Besides screening of UV-B radiation, flavonoids play key role as antioxidative agent to scavenge reactive oxygen species. Flavonoids accumulations are very much helpful in protecting the photosynthetic machineries of mesophyll tissues by epidermal screening of UV-B.Also, various other antioxidants and stress induced enzymes are synthesised against UV-B (Jordan 1996). To protect biomembranes, enhanced amounts of polyamines are produced, especially at high photosynthetically active radiation levels, increasing the stability of the biomembranes so that UV-B damage is restricted (Jordan 1996).

Plants are equipped with several mechanisms for repairing DNA. During photoreactivation, monomerization of dimers by photolyases takes place. Related enzymes are activated by high photosynthetically active radiation levels, partly explaining the mitigating effect of high photosynthetically active radiation levels on UV-B damage (Day and Neale 2002). With excision repair mechanisms, the damaged portion of DNA is detached and replaced by the new replicated DNA (Britt 1996). The parameters affecting UV-B attenuance and photomorphogenesis such as plant height, UV-B absorbing pigments, number of leaves, yield and total biomass, which reflect UV-B adaptation effects, showed more pronounced differences between cultivars (Table 5.1). The epicuticular layer of wax operates as boundary between the interior of leaf and environment, and it is an important surface character that responds to environmental stresses (Kakani et al. 2003b). About 10 to 30 % of the UV-B radiation was reflected due to epicuticular wax deposition (Caldwell et al. 1983; Holmes 1997). High lignin and epicuticular wax content of UV-B exposed plants were the protective mechanism adopted by the plants to reduce the epidermal transmittance of UV-B (Choudhary and Agrawal 2015b; Kakani et al. 2003b; Kakani et al. 2004; Tripathi and Agrawal 2013).

5.3 Effect of Ultraviolet-B Radiation on Symbiosis Controlling Factors of Legumes

Since, the large numbers of studies reported on UV-B and leguminous plants but its effects on nitrogen metabolism and symbiosis of legumes were little explored. Very few literatures are existing regarding this aspect and here we tried to provide its overview in Table 5.2. UV-B radiation is unable to penetrate into the soil so it is apparent that its effect are not direct on symbiotic process, however changes that occur in host plants due to UV-B, is responsible for changes in symbiotic associations (Choudhary et al. 2013). Some experimental work that has been done suggested that UV-B negatively influence the symbiotic relations of plants and microsymbionts. Singh (1997) showed the reduction in nitrogenase activity, nodulation and nodule diameter was generally due to reduced photosynthesis after elevated UV-B exposure. Lou et al. (2011) carried the field study with wheat and found the microbial activities shuffled towards the nonrhizospheric zones which resulted low microbial biomass carbon and nitrogen in the rhizosphere. The results were in agreement with the study conducted by Choudhary et al. (2013) in mung bean plants, and have correlated it with the increments in phenol and flavonoids contents of the roots, which affected the composition of root exudates leading to loss in symbiotic association as nodulation process was inhibited under elevated UV-B. These compounds are antimicrobial in nature and acts against root-feeding insects and microbes (Ndakidemi and Dakora 2003). Their stimulations are thus responsible for the decrease of microbial population in the rhizosphere. Roots of plants exude more momilactone-B under UV-B, which also acts as microbial depressant (Noguchi et al. 2007). Choudhary et al. (2013) have confirmed that the soil enzyme activities of mung bean rhizosphere were also negatively affected under enhanced UV-B. Enzymatic activities of the soil are key regulators to nutrient availability and microbial growth and thus have been utilized widely as soil fertility and stress indicators. Soil enzymes are used as soil health indicators because they reflect past biological activities and capability of soil for stabilizing soil organic matter. Therefore, they might be useful for detection of management effects on soils (Roldán et al. 2005; Garcıá-Ruiz et al. 2008). However, UV-B exclusion study conducted by Chouhan et al. (2008) showed the significant increment in nodulation, nodule size and leghemoglobin content, suggesting that UV-B is dangerous for nodulation and the key component of nodule which is responsible for nitrogenase activity of root nodules. Chimphango et al. (2004) concluded that 15 and 25 % of stratospheric O3 depletion had negligible impact on eight African commercial legumes however; they found decreased nitrogen fixation and nodule activity of Cyclopia maculate. Some studies conducted in green house chambers and field conditions at low UV-B dose showed that the nitrogenase activity and nodulation increased significantly (Table 5.2).

Table 5.2 Effects of UV-B radiation on symbiotic associations of leguminous plants leading to alteration in nodulation, nitrite and nitrate reductase, nitrogenase, leghaemoglobin content, root biomass and microbial biomass under different experimental conditions
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Reduction in arbuscular mycorrhizal fungus infection was observed in Calmagrostis epigeous and Carex arenaria (Van de Staaij et al. 1999, 2001) and Acer saccharum (Klironomos and Allen 1995). The important effect of high UV-B levels on arbuscular mycorrhizal fungus infection is manifested by decreased number of arbuscules in the roots of host plant. Arbuscules are responsible for nutrients exchange between the partners; reduction in their number indicates restriction in the nutrient flow. The uptake of nitrogen (Tobar et al. 1994), phosphorus (Scheiner et al. 1996; Dickson et al. 1999) and water (Rozema et al. 1986; Subramanian et al. 1995) from soil is improved by arbuscular mycorrhizal associations which were disturbed under UV-B. Enhanced UV-B influences arbuscular mycorrhizal associations via differences in UV-B absorbing flavonoids in above and below ground host plant tissues. More generally, UV-B induced the biosynthesis of polyphenolics e.g. tannins and lignin, which may possibly reduce the development of micro-organisms including arbuscular mycorrhizal fungus (Van de Staaij et al. 2001). Another reason might be that auxins level disturbed, generally UV-B reduces the levels of these phytohormones in plants (Ros and Tevini 1995). Therefore elevated UV-B may indirectly affect arbuscular mycorrhizal fungus and rhizobial associations through effects on phytohormone balance in host plant (Van de Staaij et al. 2001).

UV-B has no direct effect on belowground symbiosis, since UV-B is unable to pierce the ground. Hence, the changes in aboveground plants caused by UV-B are harmful for establishment of symbiotic associationship between the leguminous roots, arbuscular mycorrhizal fungus and rhizobia. Present review, summarizes the important factors that are affected by UV-B and are responsible directly or indirectly for establishing the symbiotic association.

5.3.1 UV-B Perception, Signal Transduction and Gene Expression

UV-B causes varied and precise responses related to gene regulation at different doses (Frohnmeyer and Staiger 2003). High UV-B exposure causes necrosis of tissues and also induces expression of stress related genes. UV-B exposure negatively affects synthesis and expression of proteins responsible for photosynthesis, like chlorophyll a/b-binding proteins (Lhcb), D1 polypeptide of PS II (psbA), Rubisco, etc. (Jordan 1996). Low UV-B radiation is helpful in promotion and expression of genes responsible for defense against UV-B (Frohnmeyer and Staiger 2003; Ulm et al. 2004). Several studies reported the genes expression for protective mechanisms, like DNA repair, reactive oxygen species detoxification and production of pigments such as flavonoids (Brosche and Strid 2003; Stratmann 2003). Enzymes related with phenylpropanoid pathway get up-regulated under UV-B and mainly responsible for flavonoid biosynthesis and other protecting compounds. Many other defense genes also ‘switched on’ under UV-B (Jordan 2002). Therefore, understanding of mechanisms linked to UV-B perception, gene regulation and signal transduction in plants are very important. However, remarkably little knowledge is available about the plants perception or signal-transduction through which UV-B regulates gene expression.

Plants have different receptors for diverse wavelength of solar light and specific receptor triggers for multiple responses related to developments in plants. Phytochrome, an important photoreceptor, initiates the pathway for signal transduction in gene regulation (Agrawal et al. 2009). Other important participants of signal transduction pathway are calmodulin, G-protein and cGMP. However, photo-morphogenetic responses on plants are mediated by various known photoreceptors; cryptochrome, phytochrome and phototropin (Frohnmeyer and Staiger 2003; Agrawal et al. 2009). These are specific receptors i.e. phytochromes mediate responses to red or far-red light, cryptochromes and phototropins mediate responses to UV-A or blue light. Photoreception systems mediated UV-B responses may be occurred by novel class photoreceptor but specifically these are yet unidentified. Highly energetic UV-B photons are also absorbed by chlorophylls, carotenoids and quinones. UV-B specific initiation of several UVB-responsive genes is regulated during transcription, the product of which can affect expression of target gene (Agrawal et al. 2009; Jordan 1996).

5.3.2 Photomorphogenic Response

Commonly UV-B can provoke photomorphogenic responses, i.e. various modifications in plant forms through radiation (Tevini and Teramura 1989; Jansen 2002). The process of light regulated plant growth is described as photomorphogenesis (Kendrick et al. 1997). Several processes are regulated during light signals that include changes to structure and form, like germination of seeds, stem elongation, expansion of leaves, pigment synthesis and initiation of flowers etc. Most of these responses are adaptive in nature. Some of these morphogenic changes may diminish the exposure of cells or leaves to UV-B exposure. In emerging seedlings it was hypothesized that, hypocotyl elongation inhibited to minimize exposure of UV-B, until UV screening pigments has been accumulated (Ballare et al. 1995). Thickening of leaves is also protective mechanism adapted by the plants due to UV-B exposure (Cen and Bornman 1993). Similarly, the loss in apical dominance (like decreased shoot length and increased axillary branching), decreased leaf expansion and increased leaf curling are plant strategies to skip direct exposure of UV-B (Barnes et al. 1996).

Photomorphogenic UV-B signaling is mediated through UV-B-specific component i.e. UV Resistance Locus8. UV Resistance Locus8 and Constitutive Photomorphogenesis1 are essential in UV-B induced expression of Elongated Hypocotyl5, which plays key role during regulation of genes concerned in photomorphogenic UV-B responses (Jenkins 2009, Oravecz et al. 2006, Singh et al. 2014).

5.3.3 Growth

Negative impacts of UV-B on growth of plants include reduction in leaf area; shoot elongation, biomass and productivity. Reduction in various growth parameters and accumulation of biomass under UV-B has been revealed by several studies conducted under natural field conditions and growth chambers (Feng et al. 2003; Kakani et al. 2003b; Searles et al. 2001; Mishra and Agrawal 2006; Singh et al. 2009). Inhibitory responses of UV-B regarding growth and development studied widely on wheat cultivars (Agrawal et al. 2004; Li et al. 2000), 20 soybean cultivars (Li et al. 2002), maize (Correia et al. 1998), cotton (Kakani et al. 2003a, b), kidney bean (Singh et al. 2014), buckwheat (Regvar et al. 2012; Yao et al. 2006a), okra (Kumari et al. 2009), pea (Agrawal and Mishra 2009) etc. Retardation in growth was resulted due to destruction of indole acetic acid (Tevini and Teramura 1989; Hopkins et al. 2002). Enhanced oxidative stress was also important factor for reduced plants growth and development under UV-B (Jansen et al. 1998). UV-B irradiation affects the light induced protein kinase which is the key regulator of plant development including growth, expansion of leaves and synthesis of pigments (Stratmann et al. 2000). Several studies on plant-UV-B response state that inhibition of whole-plant growth correlates with reduction in leaf expansion, which is more sensitive to UV-B than photosynthesis. Caldwell and Flint (1994) showed that morphological changes and reduced growth were more common responses against UV-B than those of reduced photosynthesis. Under elevated UV-B, decline in photosynthetic productivity is related to reduced plants ability to interrupt light i.e. smaller leaf area, but not with the inhibition in photosynthetic capability (Allen et al. 1998).

UV-B studies performed on growth response of legumes have revealed inter as well as intra-species variations in magnitude and type of response against UV-B might depends on experimental circumstances and UV-B intensity. A detail data on several studies concerned with UV-B mediated changes on growth and anatomical modifications in legumes are provided in Table 5.1. These changes directly or indirectly affect the symbiosis process of the leguminous plants.

5.3.4 Leaf Ultra Structure and Anatomical Changes

At the structural level, increased thickening of epidermal layer is commonly observed adaptive response against UV-B (Nagel et al. 1998; Liu et al. 2005). Increases in the cell number and spongy and palisade tissues influence the penetration of UV through the leaf (Caldwell et al. 1995). Increased thickness of leaf assessed through increase in specific leaf weight, alleviating the transmittance of UV-B penetration to inner target tissues (Caldwell et al. 1983; Kakani et al. 2003b; Singh et al. 2011; Tevini and Teramura 1989). Increased thickness of leaves facilitates plants to mitigate UV-B radiation and protects palisade tissues (Newsham et al. 1999). Thickness of leaves is associated with upper epidermis, spongy parenchyma and also spongy intercellular space (Kakani et al. 2003b). UV-induced changes to ultrastructure of birch indicated that change in leaf thickness was unable to provide complete protection to palisade layer against UV-B (Kostina et al. 2001). Santos et al. (2004) reported no change in structural integrity of leaf cells in potato by UV-B exposure. They observed changes only at utrastructural level that includes appearance of paracrystalline inclusions in peroxisome, constricted plastids and increased number of thylakoids.

Leaf expansion and anatomical development are altered by UV-B in several agronomic and tree species. Diversified anatomical responses of UV-B are reported in several leguminous plants (Table 5.1). For example, UV-B radiation caused increased leaf thickness in Brassica napus (Cen and Bornman 1993) while, UV-B exclusion showed that Petunia leaves were thicker as compared to UV-B irradiated plants (Staxen and Bornman 1994). Elevated UV-B application to sweetgum (Liquidambar styraciflua) seedlings causes no significant effect on leaf size but leaf elongation rates and area of leaf were quiet slower when exposed to 3 kJ UV-B daily (Dillenburg et al. 1995). Kostina et al. (2001) observed ultrastructural changes along with numerous lipid bodies in birch seedling (Betula pendula) when irradiated with UV-B. Similar menifestation were also found in a glasshouse by Wulff et al. (1999). The lipid accumulations may indicate accelerated cell senescence at higher UV-B irradiance (Wulff et al. 1996). Damaging effect might be the consequence of disruption of thylakoid membrane (increase thylakoid swelling, dilation of thylakoid membranes) which may breaks its structural contact with stroma, disrupt the integrity of membranes (He et al. 1994; Kakani et 2003b; Kostina et al. 2001).

5.3.5 Physiological Changes in Response to UV-B

Physiological damage like reduced photosynthesis is the common phenomena observed in various sensitive species (Choudhary and Agrawal 2014a, 2015a; Sullivan and Teramura 1989; Ziska et al. 1992; Singh et al. 2013). UV-B Damages PS II (Bornman 1989) along with stomatal limitations (Teramura 1983). Reduced photosynthesis under UV-B is directly disturbing the symbiotic relationship via low carbon supply to the symbionts. Here, we will discuss UV-B effects that mainly lead to loss in photosynthesis.

5.3.5.1 Photosynthesis

Reduction in rate of photosynthesis under enhanced UV-B revealed the impairment of PSII reactions, Rubisco activity, enzymatic processes of Calvin cycle, ATPase activity, stomatal regulation (Surabhi et al. 2009). Photoinhibition and photodamage are common phenomena against stress conditions like UV-B (Laposi et al. 2009). Concerning UV-B effects on photosynthesis, variability depends mainly on the sensitivity of plants and growth conditions. Several studies showed that UV-B caused inhibition in photosynthesis were only depicted at high intensity of UV-B (Bassman et al. 2002; Keiller and Holmes 2001). However, studies conducted simulating 15–25 % ozone depletion and 30 % increments of UV-B, observed no major changes in growth and photosynthesis of all crops including legumes (Allen et al. 1998; Chimphango et al. 2003a, b; Sullivan et al. 2003). Damaging effects on photosynthesis is widely reported in pea (Nogues and Baker 1995), cotton (Kakani et al. 2003b; Zhao et al. 2005; Kataria et al. 2012) and rapeseed (Allen et al. 1997; Sangtarash et al. 2009). Influence on photosynthesis showed inconsistent trend depending upon species, cultivars, growth environments, UV-B intensity and exposure duration (Kakani et al. 2003a; Rozema et al. 1997).

5.3.5.2 Damage to Chloroplast

Chloroplast is the cell organelle responsible for photosynthesis and anabolism of chlorophyll. Structure of chloroplast also altered after UV-B treatment, revealed by damaged chloroplast envelope, expansion of thylakoid and disintegration of lamellar system. Damage to chloroplast envelope resulted in disorganization of granal and stromal thylakoids. Peroxidative stress induced by reactive oxygen species under UV-B, leads to destruction of thylakoid membrane in chloroplast, causing damages to chlorophyll proteins and induces the breakdown of chlorophylls (Zu et al. 2004). He et al. (1994) reported changes in ultrastructure of pea chloroplast under UV-B treatment like unbalanced lamellar system and envelope disruptions. Santos et al. (2004) reported only minor ultra structural changes such as reduction in size of guard cells, changes in palisade cells and epidermal layer against UV-B in potato, however structure of chloroplasts was not affected.

5.3.5.3 Stomatal Regulation

Increased in UV-B levels tend to reduce the capacity of stomata to open under high irradiance, and its capability to close in dark (Keiller and Holmes 2001). Enhanced UV-B irradiance may affect photosynthetic efficiency negatively, by altering the stomatal conductance and thus permitting less CO2 to enter into plant cells (Zheng et al. 1996; Yang et al. 2000). In soybean reduction of stomata density and its function, transpiration rate, water use efficiency and carboxylation efficiency of plant leaves were observed under enhanced UV-B (Gitz et al. 2005). Affects of UV-B on stomatal opening and closing is through inhibition of K+ influx and by impairing ATPase proton pumps in stomatal guard cells (Wright and Murphy 1982). Contrary to this, Jansen and Van den Noort (2000) reported that high fluence rate of UV-B either stimulates stomatal opening or closing depending upon metabolic stage of guard cells. However, UV-B effect on stomatal regulation was not a major limitation for CO2 assimilation in leaf photosynthesis (Zhao et al. 2003).

5.3.5.4 Photosystems

UV-B driven inactivation of photosystem reactions have occurred predominately by inhibition to photosystem II, efficiency because PSII appeared to be most sensitive target of UV-B (Kulandaivelu and Lingakumar 2000). PSII is a complex of protein and pigments and the core is made of D1 and D2 proteins (Barber et al. 1997). Degradation of D1 and D2 proteins might be also possible at low UV-B fluence rates (Jansen et al. 1998). A complete examination of PS II revealed that both donor and acceptor sites of PS II was affected by UV-B (Melis et al. 1992). Rodrigues et al. (2006) reported that plastosemiquinones are photosensitizers for UV-B and its absorption by quinones leads to damage of PS II. UV-B driven decline in activity of PSII finally corresponds to net inhibition in CO2 assimilation during photosynthesis (Krause et al. 2003).

5.3.5.5 Calvin Cycle

Photo oxidative stress conditions under UV-B could potentially disturb CO2 fixation and reduce the content and activity of NADP+ and ribulose bisphosphate (Rubisco), as important key regulatory enzymes responsible for regeneration of ribulose bisphosphate and NADP+ as “GAPDH” and “phosphoglycerate kinase” reduced under UV-B (Xu 2008). Both these Calvin cycle enzymes are extremely sensitive against reactive oxygen species particularly H2O2 (Kaiser 1979). Consequently electron transfer is reduced, leads to singlet oxygen and superoxide radical formation (Krause 1996). Izaguirre et al. (2003) also found that some genes related to Calvin cycle enzymes were down-regulated on solar UV-B exposure. Several studies indicated that inhibition in photosynthesis is linked with enzymatic reactions during Calvin cycle under UV-B (Allen et al. 1998; Keiller and Holmes 2001; Xiong and Day 2001).

5.3.5.6 Rubisco and ATPase Activity

Modifications in Rubisco activity and its content have been identified as possible limiting factors affecting photosynthesis. Inhibition of CO2 assimilation under UV-B was resulted due to synthesis, degradation and activity of Rubisco as in Oryza sativa (Huang et al. 1993), Populus deltoids (Bassman et al. 2001) and Glycine max (Feng et al. 2003). Allen et al. (1997) reported that decrease in rate of CO2 assimilation was outcome of reduced Rubisco content and its activity. Tekeuchi et al. (2002) have also found the suppression of Rubisco synthesis during the leaf development in UV sensitive Norin 1 cultivar of rice. The action spectrum for inactivation of plasma membrane bound ATPase peaks at 290 nm, showing their sensitivity towards enhanced UV-B (Caldwell 1993). Inactivated ATPase might be resulted from damage to tryptophan through singlet oxygen (Stapleton 1992).

5.3.6 Plant Hormones

Plant hormones are signalling molecules formed in the plants and present in very low concentrations. Hormones can regulate various cellular processes locally in the targeted cells and also to other sites in the plants. Beside their main role in growth, development, flowering, leaves shedding, ripening of the fruits, etc. in the plants, they also have major role during symbiotic association of plants. UV-B is disturbing the hormonal balance of plants which indirectly affects to symbiotic associations below ground.

5.3.6.1 Abscisic Acid

Abscisic acid is well known to act as inhibitor of nodule development, as its application on Pisum sativum led to decrease in nodule number (Philips 1971). Another study on super nodulating mutant line NOD 1-3 of glycine max showed reductions in number of nodule and also dry weights (Cho and Harper 1993). Bano and Harper (2002) determined that both nodule initiation development and functioning were inhibited by abscisic acid in wild type and NOD 1-3. Philips (1971) revealed that abscisic acid may act as inhibitory to cytokinin stimulated divisions of cortical cells responsible for nodule formation suggesting the abscisic acid-cytokinin signaling interaction. Abscisic acid and cytokinin is engaged during plant development by root-shoot signaling (Davies and Zhang 1991) and symbiotic photosynthesis gas exchange (Goicoechea et al. 1997). Ratio of these two hormones positively correlated to nodule suppression and auto regulation (Caba et al. 2000; Bano et al. 2002). Bano et al. (2002) showed possible influences of plant abscisic acid and zeatin riboside ratios in nodule auto regulation. Inoculation induces reduction in abscisic acid and zeatin riboside ratio of xylem. These authors shown that ratio of hormones are deflected to leaves and promote biosynthesis of abscisic acid. After that, increased abscisic acid moves via phloem to roots, and further inhibits nodule formation. This pathway is effectively non functional in super nodulating mutants as the decrease in abscisic acid and zeatin riboside ratio of xylem did not take place and thus proper nodule number is not achieved (Bano et al. 2002). Caba et al. (2000) showed that augmentation in abscisic acid concentration of root was missing in the proposed model.

Increased amount of abscisic acid are detected in soybean roots, shoots and nodules bearing arbuscular mycorrhizal associations as compared to nodulated non mycorrhizal plants, which suggests that mycorrhizal associations contribute for abscisic acid pool in the partner host including nodule (Murakami-Mizukami et al. 1991). This might results to the activation of carbohydrate sink through abscisic acid during the seed filling phase of soybean (Murakami-Mizukami et al. 1991). They hypothesized that nodule abscisic acid increase might responsible to stimulate a analogous carbohydrate sink in root nodule and play inhibitory role. Abscisic acid might be the factor in photosynthates allocation to the site of symbiosis for symbiosomes which they utilized as energy source for respiration, nitrogen fixation and phosphorus uptake from the soil etc. However, nitrogenase activity was decreased with rising levels of endogenous abscisic acid in different plants (Dangar and Basu 1984, 1987). Similarly the daily application of abscisic acid significantly reduced nitrogen fixation capacity of pea (Gonzalez et al. 2001). It might possible that abscisic acid plays dual role in nodule development, one in negative regulation of number and second in positively regulating the development and growth of individual nodules (Furgusion and Mathesius 2003). Charbonneu and Newcomb (1985) reported level of abscisic acid in pea nodule was high in first 2 weeks of nodule development followed by the 2 week decline and then a secondary phase of elevated abscisic acid. It might be possible that the first rise in abscisic acid is related to modulation of nodule growth and number, the decline corresponds to nitrogen fixation period and the second role is related to nodule senescence (Furgusion and Mathesius 2003).

According to Fig. 5.2, UV-B might effects levels of cytokinin in plants through which the cytokinins and abscisic acid ratios get altered. Further, abscisic acid – cytokinin signaling in plants get interrupted and the establishment of symbiotic associations also get interrupted. Since, root abscisic acid and shoot abscisic acid contents are normally responsible for sucrose availability needed for the symbiotic interactions.

Fig. 5.2
figure 2

An overview of UV-B effects on symbiosis controlling factors of leguminous plants. UV-B leads to alteration in oxidative stress, photomorphogenic responses, oxidative defence system, secondary compounds, photosynthesis, growth, ultrastructure and anatomical structures of legumes. These changes in leguminous plants against UV-B are causing adverse effects to symbiotic relationship directly or indirectly. ROS reactive oxygen species, PS I photosystem I, PS II photosystem II, ABA abscisic acid

Full size image

5.3.6.2 Auxin

Auxins are important for cell division, differentiation and formation of vascular bundle. These processes are also repeated during nodule formation. Mainly this hormone is synthesized in shoot and translocated to root via transport process. Auxin import protein (AUX 1) and auxin export protein (PIN 1 and PIN 2/AGR/EIR 1) are mainly responsible for transportation to plant cells (Muday and Delong 2001). Compared to roots, nodules have high auxin content in various leguminous plants like P. sativum (Badenoch-Jones et al. 1984), P. vulgaris (Federova et al. 2000) and A.glutinosa (wheeler et al. 1979). Increased levels of auxin in legume nodules, and nodule like structures of non-legumes is also revealed by synthetic auxin 2, 4 dichlorophenoxy acetic acid (2,4-D) application (Ridge et al. 1992). Auxin: cytokinin ratio of roots is also responsible for the division of cortical cells and nodule formation (Libbenga et al. 1973). In hypernodulating nts386 mutant of soybean, the auxin-cytokinin equilibrium was observed to be lower as compared to wild type, signifying that auxin-cytokinin ratio is key regulator during nodulation (Caba et al. 2000). Labeled auxin study in Vicia sativa roots revealed inhibition in acropetal transport of auxin by rhizobia (Boot et al. 1999). Auxins also plays dual role for nodulation, during early stages, inhibition in auxin transport might resulted in the reduced auxin:cytokinin ratio, which allow to start cell divisions. Later cell divisions are suppressed due to ideal auxin levels (Furgusion and Mathesius 2003).

Since, auxins strongly absorbs UV-B part of spectrum, therefore increased UV-B might reduced auxin levels (Ros and Tevini 1995). Thus makes the changes in auxin:cytokinin ratio and thus creating alterations in root shoot communications negatively and influencing the symbiotic interactions. Therefore, shifting ratio of these hormones may cause decreased nodulation (Baron and Zambryski 1995; Hirsch and Fang 1994). Since the auxin: cytokinin ratio is responsible for initiation of cell cycle during which divisions of pericycle and cortical cell occurs in symbiotic interactions, hence UV-B negatively influence and creates alterations in symbiotic interactions (Fig. 5.2).

5.3.6.3 Cytokinin

Cytokinin is the plant hormones having key role for activation of cell cycle. Cell cycle initiates formation of nodule primordium (Foucher and Kondorosi 2000, Yang et al. 1994). Cytokinin with auxin and ethylene is important for the progression of cell cycle in plants (D Agostino and Keiber 1999). Pseudo-nodule structure have been observed due to post application of cytokinin various leguminous and nonleguminous plants including Nicotiana tabaccum (Arora et al. 1959), A. glutinosa (Reedriguez-Barrueco and Burmudez de Castro 1973) , Macroptelium acropurpurium (Relic et al. 1993), pea (Libbenga et al. 1973), Medicago sativa (Baeur et al. 1996; Cooper and Long 1994). Concentration of cytokinin is responsible for its stimulatory or inhibitory effects on nodulation (Lorteau et al. 2001). Role of cytokinins during nodule developments includes activation of genes and cell cycle (Jelenska et al. 2000). Molecular markers screening in Medicago sativa recognized seven nodulin genes regulated by the cytokinins and four of which also induced by the auxin suggesting partial overlaps between auxin and cytokinin regulated pathways during nodulation (Jimenez-zurdo et al. 2000). Cytokinin plays an important function in carbohydrate sink for developing nodule, as they induces formation of starch in root cortex as noticed during rhizobium infection (Baeur et al. 1996).

Since increased UV-B affects the cytokinin signaling by altering the auxin:cytokinin ratio and cytokinin:abscisic acid ratio and these ratios of hormones play a very important role throughout the symbiotic associationship with rhizobia and arbuscular mycorrhiza. Cytokinin-abscisic acid is mandatory for the sucrose availability, and auxin-cytokinin important for cell cycle activation.

5.3.6.4 Gibberellic Acid

Not much information is present regarding the signaling of gibberellic acid during symbiotic associations. However, decline in nodulation were noticed after gibberellic acid application to plants in various studies (Galston 1959; Mes 1959; Thurber et al. 1958). Dullart and Duba (1970) reported stimulated auxin production from L-tryptophan in nodule extracts after gibberellic acid application in L. luteus. They also suggested that signaling interactions existed, where gibberellic acid was responsible to increase the bioproduction or decrease the metabolism of auxin (Fig. 5.2). Whereas, reverse interaction was confirmed in stems where gibberellic acid requires auxin for its biosynthesis (Ross et al. 2000). Auxin was shown to promote root growth in Arabidopsis by modulating cellular responses to gibberellic acid (Fu and Harberd 2003). It might be possible that similar interactions involved between these hormones for regulating symbiotic interactions. Gibberellic acid promotes α-amylase production (Gubler et al. 1995), involved in starch metabolism. Several evidences are present regarding this in various fungal species (Rademacher 2000).

Gibberellic acid, α-amylase and starch can be correlated as the gibberellic acid stimulates synthesis of α-amylase then synthesizes sugar which is utilized by the symbiosomes during symbiotic interactions. Alteration in auxins under UV-B is responsible for disturbances in signaling interactions of gibberellic acid and auxin, might be influencing the symbiotic relationship (Fig. 5.2).

5.3.6.5 Salicylic Acid

Salicylic acid is the important vital components of signalling pathways responsible for the induction of defense mechanism (Creelman et al. 1997; Durner et al. 1997). Salicylic acid plays key role during systemic acquired resistance and it induces the genes encoding pathogen-related proteins. More formation of salicylic acid under elevated UV-B has been observed in various plants (Choudhary and Agrawal 2014a, b; Sharma et al. 1996). More salicylic acid accumulation leads to higher oxidative stress, and it may also leads to plants death (Horvath et al. 2007). The oxidative stress caused by salicylic acid was due to its inhibitory function on activities of antioxidative enzymes such as catalase and ascorbate peroxidase (Durner and Klessig 1995; Rao et al. 1997; Vicente and Plasencia 2011), which led to higher H2O2 accumulation.

5.3.6.6 Jasmonic Acid

Jasmonic acid is also vital components of signalling pathways responsible for the activation of defense mechanism (Creelman et al. 1997; Durner et al. 1997). Responses of plants to biotic or abiotic stresses are coordinated through jasmonic acid locally as well as systemically. Jasmonic acid and its derivatives like methyl jasmonate are mainly responsible for gene induction related with defense during wounding or pathogens attack. An increase of jasmonic acid concentrations was reported under enhanced UV-B in various plants like mung bean (Choudhary and Agrawal 2014a) and pea (Choudhary and Agrawal 2014b). Beside the role in defence mechanism of plants jasmonic acid is also important for induction of nod genes (Rosas et al. 1998).

5.3.7 Flavonoids

Rhizobia react on root exudates of host plant depending on its species, this can be host specific or more general one (Tok et al. 1997). Root exudates of plants are mainly composed of flavonoids i.e. isoflavones and flavones (Stafford 1997). Similarly, flavonoids secreted via root exudates play an essential role during plant-mycorrhizal symbiosis. Excreted flavonoids have clear effect on fungus (Sequeira et al. 1991) as daidzein stimulated spore germination, myricetin and quercetin increases hyphal growth in germinated spores, when quercetin concentration increases it becomes inhibiting factor for hyphal growth (Chabot et al. 1992). However, flavonoids are not essential in all the cases because maize plants lacking essential enzymes responsible for flavonoid biosynthesis, showed normal mycorrhizal growth (Shirley 1996). Flavonoids have numerous roles during plant development, defence and symbiotic interactions (Dakora 1995) Flavonoids also protects dividing cells to oxidative damage, as they also have antioxidative properties (Rice-Evans and Miller 1996).

Changes in flavonoid composition present in root exudates of host plant after UV-B irradiation leads to less effective nod gene stimulus since a very specific flavonid composition is necessary for induction of nod gene (Tok et al. 1997). Irradiation with UV-B leads to increased synthesis of phenylpropanoid pathway enzymes i.e. phenylammonia lyase and chalcone synthase. PAL and CHS are essential for the biosynthesis of flavonoids (Meijkamp et al. 1999). These enzymes might be translocated from top of shoot to root and may disturb the composition of root exudates. A change in root exudates under UV-B is also responsible for reduced nodule size and number.

5.4 Conclusion

Several studies carried with leguminous plants have reported harmful effects of UV-B. The great agricultural and environmental significance of legumes is their capacity to establish symbiosis with rhizobia which makes legumes a target crop in sustainable agriculture. The healthy tripartite association of legume-rhizobia-mycorrhiza is marvelous for maintaining the yield of crops vis-à-vis the fertility of soil. This association is also good for rehabilitation of badly ruined lands and desertified habitats. Using beneficial microbes in agronomic practices is promising and sustainable low-input soil management ventures. Therefore, more researches are required on this field exploring the legume rhizosphere and to identify the microbes suffering from the harmful impact of UV-B. After identifying microbes, it is suggested for their cultivation at larger scale and must be added in the soil as biofertilizers as per need during cultivation of pulse crops in areas facing the risk of high UV-B.