Importance of Legumes and Role of Sulphur Oxidizing Bacteria for Their Production: A Review

Legumes are rich source of valuable food proteins. Grain legumes, being a relatively cheap, non-animal, environment friendly, has played an important role as a source of proteins, micro-nutrients and numerous vitamins in human nutrition for many years (Pingoliya et al., 2014). Nutritionally important legumes such as chickpea, mungbean, pigeonpea, cowpea, lentils, blackgram, soybean and peanut etc. have been the part of diet for millions of Asians, Africans, Europeans, Americans and Kangaroos. The legumes are used as food in various forms as such or after processing into many varieties (Foyer et al., 2016). Apart from food, they have enormous capability of fixing nitrogen biologically with the help of their symbiotic partners (rhizobia), thus improving the atmosphere and soil by decreasing dependency on chemical fertilizers (Ingram, 2017). We are lagging behind in sufficient production of legumes according to our increasing population due to negligence (Foyer et al., 2016), less genetic improvement of efficient legume varieties and continuously depleting nutrients like N, P, S, K etc. from soil (Morris, 2003).
       
Sulphur is a crucial nutrient for plants along with N, P, K, as it is integral constituent of amino acids, proteins, metal cofactors, vitamins, coenzymes and a large number of secondary metabolites. Sulphur deficiency decreases plant growth, photosynthesis and seed yield in both legumes and non-legumes. Addition of sulphur to nodulated legumes, not only helps in synthesis of S-containing amino acids but also increase the amount of N in leaves and stems and amounts of N fixed in the soil (Becana et al., 2018; Zhao et al., 1999). The sulphur content of the atmosphere is very small (1.8 million tons) than hydrosphere and pedosphere and generates from abiotic (natural and anthropogenic) and biotic sources (Kelly et al., 1990). The consumption and demand of sulphur is progressively increasing day by day, year after year globally (Halfawi et al., 2010).
       
Most of the soil around the world now a days are facing the sulphur deficiency problem due to multiple reasons including use of sulphur free fertilizers, intensive agriculture growing high yielding varieties and very less sulphur returns with organic manures (Jamal et al., 2010). Also the sulphur compounds present in the soil, is found in unavailable or bounded form, which can’t be up taken by plants including legumes. Sulphur oxidizing bacteria or sulphur biofertilizers are microbial preparation of sulphur oxidizing microbes containing more than 0.5 × 10  cells/ml of active strains having significant role in equipping better rhizosphere for plant growth, improving soil fertility, providing minerals, substituting chemical pesticides, reducing production costs and environmental pollution (Halfawi et al., 2010).
 
Applications of legumes
 
There are variety of legume grains, which are classified into pulses and non-pulses by FAO (FAOSAT data, 2004). Pulses are further of various type; mung bean/green gram (Vigna radiate), lima bean (Vigna lunatus), kidney bean (Phaseolus vulgaris), adzuki bean (Vigna angularis), black gram/urd (Vigna mungo), rice bean (Vigna umbellate), scarlet runner bean (Phaseolus coccineus), Moth bean (Vigna acontifolia), Tepary bean (Phaseolus acutifolius) etc. comes under dry beans (Phaseolus spp. including several species now in Vigna). Horse bean, broad bean and field beans are placed under dry broad beans (Vicia faba) category. Third category is of dry peas (Pisum spp.) including protein pea (Pisum sativum var. arvense) and garden pea (Pisum sativum var. sativum). Some other pulses are pigeon pea (Cajanus cajan), lentils (Lens culinaris), dry cowpea/blackeye pea (Vigna unguiculata ssp. Dekindtiana), chickpea (Cicer arietinum), bambara groundnut (Vigna subterranean), vetch (Vicia sativa) and lupins (Lupinus spp.). Among minor pulses are jack bean (Canavalia ensiformis), lablab/hyacinth bean (Lablab purpureus), velvet bean (Mucuna pruriens var. utilis), winged bean (Psophocarpus teragonolobus) and yam bean (Pachyrrizus erosus) etc. peanut (Arachis hypogaea) and soybean (Glycine max) are placed under Non-pulses or oil-crops. These have broad term applications (Fig 1) in various aspects, which are discussed in details in this part of review.
 

 
An important feed for humans and livestock equally
 
The Leguminosae is one of the supreme groups of plants, which is placed at second after cereals (grasses) in supplying food to the world’s everlasting population. In rank order from higher to low, bean, pea, chickpea, broad bean, pigeon pea, cowpea and lentils (Fig 2) are the primary dietary pulses to be used as human feed (National Academy of Science, 1994). In comparison to cereals, the legumes are important food grains having high amount of proteins, essential amino acids, vitamins and minerals content, providing a highly nutritious food diet for humans and livestock (Foyer et al., 2016; Pingoliya et al., 2014).
 

       
The evidence of using legumes as an integral part of human food habits all over the world’s continents can be seen from archaeological deposits and their mention in religious scripts and mythologies like Indian Vedas, Sumerian texts and the Egyptian hieroglyphs, proves its importance from ancient times (Hancock, 2002).
       
The proteins content of legume seeds is approximately 20% of dry weight in beans and pea and 38 to 40% in lupin (an alternative to soybean in European countries) and soybean (Guéguen and Cerletti, 1994).  Pulses also have some proteins called as storage proteins and some minor proteins like amylase and protease inhibitors, lipoxygenase, lectins and defence proteins and others affecting nutritional/functional property of the legumes (Guéguen and Cerletti, 1994).
       
Along with typical human and animal food uses, legumes can also be powdered into flour and different types of products like bread, tortillas, chips, Pop beans, doughnuts, soybean candy, spreads, in liquid form to produce milks, food supplements like veg proteins, yogurt and feed for infants formula can be prepared (Graham and Vance, 2003). Legume flours are also a good source of minerals or micronutrients like iron, magnesium, calcium, zinc, selenium, potassium, phosphorous and other trace elements and vitamins such as vit. A, E and B, B1 (thiamine), B3 and the water-soluble ones like vit. B2, B6 and B9 (folic acid) and carotenoids. Lupin (conglutin g) and other legume proteins have been reported to be having proficiency of interaction with many metallic ions; thus act as metal biological carrier and helps in their absorption (Jahreis et al., 2016; Duranti et al., 2001).
       
Only soya and peanut are responsible for providing more than 35% of the world’s treated oil from vegetables. In addition to those Legumes are used for animal’s feed also in the form of fodders, green manures and forages, e.g., Medicago (alfalfa), Lupinus (lupin) and Trifolium (clover). Soybean and peanut are good source of dietary protein for the swine or poultry industries (Graham and Vance, 2003).
 
Legumes as direct proteins for human health or an alternative to animal proteins
 
For last many years, people are attracting towards vegan nutrition as they restricting themselves from consuming animal products due to multiple benefit. Legume proteins are cheaper than animal proteins (Singh and Singh, 1992). Plant proteins helps in lowering the blood pressure and also improves lipid metabolism and so on (Fechner et al., 2014; Frost et al., 2014).
       
Almost three kg of plant protein is required to produce one kg of meat protein in animal husbandry (chicken and pork). Hence about two 2 kg of plant protein is completely lost during consumption of one kg of animal. Meier and co-workers (2014) reported that in Germany, land used for generation of food (2,365 m²/person/year) is more than what is available to the entire population (1,920 m²/person/year) to live and 69% of this land is being used for animal production only. So by consuming plant proteins directly we can save lot of energy, environment and resources including land needed for the animal production (Jahreis et al., 2016).
 
Health and Medicine
 
(i) Diabetic control by maintaining GI (Glycaemic index)
 
The dry legumes, having high indigestible fibre content, preventing insulin-resistance, small glycaemic index and the occurrence of other minor components like saponins, oligosaccharides, phytosterols etc. are leading agents for controlling diabetes in diabetic individuals (Kushi et al., 1999). Intake of grain legumes has been proved to be effective in major decrease in fasting blood glucose level, insulin thus decreasing risk of prediabetes, diabetes control including diabetes-associated complications, especially cardiovascular disease (Hashemi et al., 2015). Also the diabetic patients complications can be controlled by ameliorating oxidative stress as the high phenolic compounds (particularly procyanidin) of pulses, have high antioxidant activity for scavenging free radicals (Xu and Chang, 2012). Moreover, other factors, such as low GI, protein, fiber, vitamins and minerals like Mg have ability of lowering oxidative stress. Legume grains can also improve oxidative stress by reducing the dyslipidemia and hyperglycemia.
 
(ii) Overweight and obesity control
 
Legumes helps in maintaining a regular body weight despite of having lipids, starch and proteins due to their more satiety effect by lowering the overall daily food intake. In clinical trials, bread made from lupin enriched flour, reduced appetite and energy intake, improved glucose control, plasma lipids, the risk markers of inflammation, cardiovascular diseases and repressed plasma ghrelin (orexigenic hormone stimulating appetite) and also decreased blood pressure and insulin responses (Fechner et al., 2014; Belski et al., 2011). Pulses can also act as prebiotics thus improving bowel flora, affecting production of gut hormones and consequently appetite. Legume fibres having high soluble components, plays important role in volatile fatty acids synthesis, thus inhibiting cholesterol synthesis and butyric acid, lowers the risk of colorectal cancer (Fechner et al., 2014).
 
(iii) Effect of legumes on lipid homeostasis and hypocholesterolaemia
 
Intake of legumes in diet improves blood lipids level in patients with hypercholesterolemia consequently reduce the risk of cardiovascular disease (CVD) and acetic acid augments the feeling of satiation thus improves central homeostatic mechanisms by interacting with baroreceptors present in the gastrointestinal tract (Frost et al., 2014). Due to intake of soybean protein, plasma cholesterol level decreased up to 35% and also notable decrease of triglyceride levels was observed in rats thus reduced CVD.
 
(iv) Anti-carcinogenic and inflammatory effects of legumes
 
Various micro-components of legumes like lectins, phytosterols, protease inhibitor, phytates and saponins plays an important role in fighting against various kind of cancers by performing various activities like partial starch digestion, sinking of fermentation processes and decreased re-absorption of cholesterol. Clemente and co-workers reported in 2004, the anti-proliferative effect on human colon malignancy of recombinant wild-type Bowman-Birk inhibitors (BBI) extracted from pea seeds. Anti-inflammatory and ulcer functions of BBI and a legume herb; licorice (Glycyrrhiza glabra) has been found in Chinese medicine and others (Clemente et al., 2004; Stephens, 1997).
 
(v) Assistance in female health
 
Legumes (especially alfalfa sprouts and soybeans) contain phytoestrogens having oestrogenic and anti-oestrogenic activity, have been used to nullify menopause symptoms and to keep up bone health in women (Stephens, 1997). Isoflavonoids (extracted from soya and red clover), now used as a better substitute for hormone replacement therapy (HRT) in postmenopausal women for menopausal syndromes (Beck et al., 2003).
 
Other variety of applications
 
➢ Legumes are also being used in nutraceutics, a term coined in 1979, as the result of combined applications of  nutrients and pharmaceutics. Nutraceutical compound can be defines as “any food, or part of food having health benefits, including the prevention and treatment of disease” (Duranti, 2006).
➢ Legumes plays a bio-active roles directly or as in the form of originators of biologically active peptides in many physiological activities like immuno-modulation, anti-thrombotic, anti-hypertensive and opioid activities (Kostyra,1996). Also the chemicals present in the legumes proves beneficial as anti-allergenic, anti-viral and anti-microbial.
➢ In industries, legumes have potential use in manufacturing biodegradable plastics (Paetau et al., 1994), oils, inks, dyes, cosmetics, textile, timber, tannins and gums, (Morris, 1997). Some legume trees also provide valuable resins, used in polishes, paints and lacquers.
➢ Rotenone derived from different species of Lonchocarpus and Derris are used as an active agent (poison) against fish or molluscicide and as insecticide (Singh and Basu, 2012).
➢ In many countries like USA, legumes like soybean are being used in the production of biodiesel fuel.
➢ Research is going on in the direction of replacing fishmeal by legumes as C20-C22 fish oil is more beneficial to human health than plant’s (particularly legumes) C18 oil. Fishes require proteins (predominantly S-rich amino acids) and fatty acids, oils, or lipids in their diets but not carbohydrate (Glencross, 2000).
 
Legume production: Current scenario
 
The Leguminosae is considered as one of the biggest families of angiosperms (flowering plants) having more than 18,000-19,000 species of legumes, which are classified into 670-750 genera. They are placed on second number after the Graminiae in their prominence to humans (Polhill et al., 1981). Legumes (grain and forage) are being cultivated on around 180 million ha, or on 12-15% of the Earth’s arable land accounting for 27% of the world’s primary crop production with soybean as most yielding. Grain legumes only, contributes around 33% of the dietary protein N₂ requirements of human beings. There is comparatively very less increase in yields of legume than cereals i.e. 0-2% and hence contribute a very small share of staple food when compared with cereals. Also the increase in area of legume production in the past fifty years is very less (~4 times) than cereals. The cereal production increase is due to advance agronomic practices, use of new genomic varieties and yield increase but in case of legumes it is due to increase in area planted only. The increase in grain yields are only between 0-2% per year. In recent five decades, the cereal production all over the world, has increased by triple rate while, there is only 60% increment in legume production. This comparative low yield in legumes is due to less genetic diversity in grain legumes breeding programmes than cereals. In Africa, there is >300% yield gap for legumes including only 10-20% yield of cowpea’s genetic potential (Foyer et al., 2016).
       
Grain legume breeding programs lagging behind due to less availability of genetic diversity (Cowling et al., 2017) so that there is significant scope for legume genetic improvement, which can further help in bridging the genetic gap and unlocking valuable genes like drought and heat stress tolerance (Varshney, 2016). Recognizing the world legume potential to provide a sustainable solution to food and protein security by keeping pace with population increase, the World Summit on Food Security, 2009 put the goal of 70 per cent increase in agricultural output of legumes by 2050. For the improvement of legume breeding programmes, it is necessary for legume germplasm centres like GENESYS (Global Gateway to Genetic Resources), National Genetic Resource Centres, China, The National Institute of Agrobiological Sciences (NIAS) Genebank, Japan and National Bureau of Plant Genetic Resources (NBPGR), India etc. to have a systematic inventory of legume germplasm collection and provide easy access to breeders (GENESYS, 2016).
 
Sulphur nutrition and legumes
 
Sulphur (S) is considered as an essential nutrient along with N, P and K for legume growth and development. It is responsible for synthesis of amino acids viz., methionine, cystine, cysteine and act as disulphide bridge in proteins. Sulphur also plays important role in the synthesis of nitrogenase enzyme, thiol ring, chlorophyll molecule, lipoic acid, vitamins B, thiamine and biotin and helps in metabolism of nodule biosynthesis, N₂-fixation and carbohydrates by affecting proteolytic enzymes (Ahmed et al., 2017; Najar et al., 2011).
       
Addition of S to the legumes also increased the amount of nitrogen in stem, leaves and total content of shoot as S is direct constituent of nitrogenase enzyme and nodule formation, thus having role in N-uptake (Becana et al., 2018; Zhao et al., 1999). S-addition doubled the amount of N₂-fixed at all four stages of growth in pea and leaf chlorophyll content and shoot dry weight were also increased significantly at the flowering and pod fill stage, respectively. The roots were also having the high concentrations of S (10 mg/g) of dry weight. Thus clearly indicating a high demand for sulphur by nodules/ roots of pea and S-deficiency directly inhibit N₂-fixation. It has been also found that S-nutrition also promotes the efficiency of both nitrogenous and phosphorus fertilizers and other micronutrients uptake (Shrivastava et al., 2018; Zhao et al., 1999).
 
Sulphur containing amino acids- An important constituent of legumes
 
The content of sulphur amino acids was calculated as 3% of total protein in soya and chickpea. The sulphur content of different legume flours were found as Lupin (280±49 mg/100 g), Green pea (158 mg/100 g), Chickpea (219±14 mg/100 g) and Soya (389±25 mg/100 g). If we calculate only in terms of nutrition, all legume storage proteins are relatively low in sulphur-containing amino acids i.e. methionine and cysteine but S nutrition also directly affect nodule biosynthesis and nitrogen fixation and uptake by whole plant. Hence sulphur nutrition plays very important for legumes (Jahreis et al., 2016).
 
Emerging sulphur deficiencies
 
Sulphur has become a limiting factor for crop yield and quality in many countries including India (Zhao et al., 1999). Approximately 57 million hectares (about 41%) of total arable land in India (142 million hectares), is suffering from various degrees of sulphur insufficiency. The decrease in atmospheric-S inputs over the last 2 to 3 decades, ignorance of S-containing fertilizers like single superphosphate (SSP) and growing of high yielding varieties twice or thrice a year are main factor responsible for S-deficiency (Daemmgen et al., 1997). The characteristic symptoms shown by plants including legumes, deficient in sulphur are reduction in photosynthesis rate, nitrogen uptake and growth rate of the plant with more effects on the growth of shoots than that of roots. Generally plants deficient in sulphur become dwarf, yellow, chlorosis of younger leaves, necrosis in late stages of development and spindle with short slender stalks (Vidyalakshmi et al., 2009).
 
Sulphur oxidizing bacteria (SOB)
 
At the earliest, the sulphur bacteria were recognized by the middle half of the 19^th century with pioneers; Martinus Beijerinck and Cornelis van Niel, who studied the oxidation of sulphur in bacterial chemosynthesis and photosynthesis and the filamentous sulphur bacteria were studied by Winogradsky himself in 1887 (Kelly et al., 1997). Microorganisms having capability of oxidizing inorganic S-compounds with sulphate as an end product are recognized as sulphur oxidizing microorganisms (SOM) and if S is oxidized by bacteria then they are called as SOB. Microorganisms play a significant role in S transformations. SO₄^2- is taken up as a nutrient by plants and microbes and reduced to sulphide, which is then further assimilated into S-containing amino acids, enzymes and other organic compounds (Friedrich et al., 2001).
       
There are variety of microorganisms having capability of sulphur oxidation. Examples of SOB are Aquaspirillum sp., Acidithiobacillus sp., Aquifer sp., Beggiatoa sp., Bacillus sp., Pseudomonas sp., Paracoccus sp., Starkeya sp., Xanthobacter sp., Thiobacillus ferrooxidans, Thiobacillus thiooxidans, Chromatium sp., Allochromatium sp., Rhodovulum sulphidophilum, Rhodobacter sp., Rhodopseudomonas acidophila, Thiocaspa sp., Chlorobi sp. etc. Sulfolobus sp., Acidianus sp. are some archaea oxidizing sulphur. Among S-oxidizing fungus some important are Alternaria tenuis, Aureobasidium pullulans, Epicocum nigrum, Scolecobasidium constrictum, Penicillium sp., Myrothecium cinctum and Aspergillus sp. etc. (Chaudhary and Goyal, 2019; Sahu et al., 2018).
       
In a study done by Halfawi et al., 2010, different treatments were made in clay loam soil with sulphur oxidizing bacteria (SOB), municipal waste compost (O.M), elemental sulphur (S^o) and cabronite, taken either alone or in combination with others. The cowpea seeds were also inoculated with the specific root nodule bacteria (Okadin) before planting. There was significant increment recorded in all the treatments having S^o, S.O.B, O.M and cabronite each alone or in combination in cowpea plants. There was also a positive effect seen in the different nutrient content (S, P, K, N, Fe, Mn, Zn and Cu) in the dry weights of roots, shoots, seeds and soil as well. The application of S^o with SOB upgraded the accessibility and uptake of macro and micro nutrients along with nutrients content of the used soil (Shrivastava et al., 2018).
 
Metabolism of S in SOB
 
The sulphur present in environment is made available to plant in their usable form through Sulphur cycle (Fig 3) comprising of main four steps:
1. Firstly organic sulphur is mineralized with the help of prokaryotic organisms (SOB, Fungi and archaea).
2. Formation of sulphate (SO₄^2-) by oxidation of So, hydrogen sulphide (H₂S) and sulphide (S^2-).
3. Then this sulphate (SO₄^2-) get reduced to S^2-.
4. Finally assimilatory reduction of SO₄^2- into amino acid and other organic compounds takes place (Sahu et al., 2018).
 
                              
 
The sulphur is oxidized biologically from -2 reduced form (sulphide) of sulphur to +6 (sulphate) oxidation state through several common intermediate compounds for example, elemental sulphur (S^o), polysulphide, thiosulphate, polythionates and sulphite as, S ® S₂O₂^-3 ® S₄O₂^-6 ® SO₄^-2. These microrganisms are chemolithotrophic in nature as they meet their energy requirements from reduced form of organic and inorganic S-compounds such as H₂S, sulphide and elemental sulphur (S^o).
       
Two major pathways of thiosulphate (sulphur and sulphide) oxidation has been identified so far in SOB are; 1. Paracoccus sulfur oxidation (PSO) pathway found in Thiobacillus (Paracoccus versutus), P. denitrificans and Xanthobacter spp. etc. and 2. The ‘S4intermediate’ (S4I) pathway found in obligate chemolithotrophs (e.g. Thiobacillus tepidarius, T. ferrooxidans, T. thiooxidans etc.) and facultative species like T. acidophilus and T. aquaesulis, all of which are having tetrathionate as intermediate. There are many enzymes involved in the oxidation of sulphur out of which mostly are common to both pathways. Tetrathionate synthase (thiosulfate oxidizing enzyme), is found in S4I pathway, converts two thiosulfate molecules into tetrathionate (S₄O₆^2-). Sulphite oxidase (no need of AMP for activity), required in both pathways and for all S-oxidizing chemolithotrophs, oxidize sulphite into sulphate. One other enzyme rhodanese (a thiosulfate-S transferase) appears universal among all bacteria including SOB. Thiosulfate-oxidizing multi-enzyme system (TOMES) has been reported in Thiobacillus (Paracoccus) versutus, consisting of four components required for its activity; a thiosulphate binding enzyme (A; 16 kDa), which binds to thiosulphate and a second enzyme (B; 61 kDa) and cytochromes C551 (260 kDa) and C552:5 (56 kDa). The S-oxidation system is regulated by sox gene products (Kelly et al., 1997). Seven genes, i.e. soxXYZABCD, were found to code for proteins essential used in for sulfur oxidation mechanism in vitro (Friedrich et al., 2001).

Importance of S and SOB in improving saline soil
 
As we know saline soil slows down the plant development besides there is more requirement of energy for absorption of water and nutrient in saline soil (Stamford et al., 2002). In another study, Stamford et al., 2002 found that sulphur treated with SOB (Thiobacillus) amplified the yield of yam bean and cowpea by reducing the EC of soil saturation and pH from 8.2 to 4.7, hence improved the sodic and saline soil. They used different salts like CaCl₂, NaHCO₃, MgCl₂, NaCl and KCl and the H⁺ ion liberated due to pH reduction by formation of sulphuric acid by SOB with S resulted in dissociation of H⁺ ion by Na⁺ and other ions, enhancing its subsequent leaching, proper channelization and retention of various ions Like Na⁺, K⁺, Mg²⁺, Al³⁺, Ca²⁺ accordingly (Rupela and Tauro, 1973).
       
Ahmed et al., 2017 also reported that in treatments of wheat having S and gypsum detoxified the detrimental effects of saline soil by exchanging the Na⁺. The treatments having S and gypsum resulted in more vegetative growth due to improved physical properties of soil as the amount of nutrients like N, P, Mn, Fe and Zn were increased due to synergistic effect of SOB and S sources (Ahmed et al., 2017; Motior et al., 2011; Kayser et al., 2001).
 
Importance of sulphur in Symbiotic nitrogen fixation and nodulation
 
To increase the nitrogen fixation by legumes, a molecular understanding of mechanism, nodule formation and essential nutrients require for these is necessary. These take account of the identification of the sulphate transporter (SST1) and process of sulphur transportation in symbiosomal membrane, role of glutathione manufactured in the bacteroids and host cells for nodule function and mechanism of sulphur assimilation in legume plants, which is reprogrammed during symbiosis (Becana et al., 2018).
       
The sulphur nutrition is positively linked to symbiotic nitrogen fixation (SNF) in nodulated legumes and sulphur deficiency ultimately results into reduction of nodulation process, nodule metabolism, nitrate uptake and thus direct inhibition of symbiotic nitrogen fixation (Becana et al., 2018; Varin et al., 2010). These ill effects are due to a defect in nitrogenase synthesis, less availability of S-containing amino acids i.e. Met and Cys, thus enhancement of N-rich amino acids (arginine, asparagine and histidine) in nodulated roots and reduction in ferredoxin, leghemoglobin, ATP and glucose in nodules. Hence ample amount of sulphur to legumes strikingly increases nodulation and SNF (Varin et al., 2010). There is evidence of decrease in nodule number and mass in white clover (Trifolium repens) due to sulphur deficiency (Varin et al., 2010; Scherer, 2008).
       
The main enzyme of symbiotic nitrogen fixation, the nitrogenase, is extraordinarily rich in sulphur (contains [4Fe-4S]) suggesting the importance of S in nitrogenase biosynthesis and symbiosis. Another protein found in nodule is ferredoxin, which is a Cys-rich protein and it functions as transportation of electrons to nitrogenase (Carter et al., 1980). Cysteine and methionine are formed inside bacteroids rhizobia by a complex and active process. An important function of S in bacteroids is the sulphation of cell surface polysaccharides and nodulation (Nod) factors. The thiol group made up of sulphur also plays a significant role in catalysis, intracellular redox signalling and post-translational modifications such as oxidation to disulfide (S₂), sulfinic (-SO₂H), sulfenic (-SOH) and sulfonic (-SO₃H) acids; sulphur nitrosylation (-SNO); persulfidation (-SSH); and glutathionylation (-SS-glutathione) (Becana et al., 2018; Waszczak et al., 2015).
 
Challenges in legume cultivation and future perspectives
 
Sustainable development through improving environment
 
As we know, N is the main limiting nutrient for plants and legume cultivation. N₂-fixation can solve the problem by colonizing disturbed ecosystems through their symbiotic nitrogen fixing ability, thus provides economically sustainable advantages for farming (Vitousek et al., 1997). Near about 200 million tons of N₂ are fixed by biological nitrogen fixation annually worldwide (Mahmud et al., 2020) and in USA only, approx. 21 Mt N is being fixed by legume and rhizobium symbioses, resulting five to seven Mt N return to soils, hence saving US-8 to 12 billion dollar (Reeves et al., 2016).
       
Other than the direct beneficial effects of N₂ fixation, legumes provide multiple benefits like enhanced yield, reduced greenhouse gas emissions (Barton et al., 2014), weed, insect and pathogen control, less occurrence of disease (Duranti, 2006), improving soil stability and structure and increased nitrogen-use efficiency (Mahmud et al., 2020). Along with betterment of nitrogen use, legumes improves reach to other vital elements like phosphorus (P), K, Fe etc. Legumes also generates more income to poor farmers as they are more economic than cereals in cultivation (Kumbhare et al., 2014). Legumes also increases protein content of cereal grains and in aggregate soil organic matter, when rotated with cereals or other non-leguminous crops in farms (Foyer et al., 2016).
 
Mitigating climate change
 
The change in climate will influence legume production in some positive ways like increased levels of atmospheric CO₂ will favour legumes in gaining carbon as they use C3 photosynthesis and also higher temperature will help in faster rates of plant development, thus reducing growing season and exposure. The predicted higher temperature will upsurge production of lentil, chickpea, broad bean, dry pea, grasspea and lupin in developed countries of Northern Hemisphere such as Canada, northern Europe, France and Russia. Pea and faba beans cultivation is already increased in Finland (Ramirez et al., 2016; Peltonen et al., 2009). Although there are some constraints of climate change which are discussed as following including other challenges:
➢ Legumes can’t be cultivated on same land again and again as it can lead to formation of autotoxins, which further will  affect the soil micobiome (Huang et al., 2013).
➢ The presence of anti-nutritional compounds (ANC) in legumes is also problematic in accepting legumes in daily diet. The ANC results in the inhibition of numerous digestive enzymes and proved toxic to the growth of experimental     animals and also cause nausea, diarrhoea after injecting in humans. However, proper heat processing and soaking,     leaching, fermentation or dehulling of legumes not only result in complete elimination of these undesirable outcomes but may even play positively in nutritional role as they contain high sulphur-containing amino acids than most  of the seed proteins (Uauy et al., 1995).
➢ The low yield of legumes is due to very less commercial exploitation of all 18,000 species present, as only 65 species (50 species of forage legumes and around 15 of grain legume), are exploited out of these 18,000 species (Kelly et al., 2000). The five most constraints in this are lack of breeding acceptable quality traits, their placement  into farming systems of warm climates, dependency of legumes and rhizobia to grow at target edaphic niche and less acquisition to suitable germplasm and difficulties in market penetration of novel legumes due to high price (Nelson and Hawthorne, 2000).
➢ Developing legumes to substitute the fishmeal in aquaculture feeds is facing great challenge to managing the carbohydrate part of legumes as it can lead to decreased digestibility of feed resulting in reduced protein retention (Glencross, 2000).
➢ Continuous change in climate also put many challenge to legume production like the enhancement in decaying  of canopy, photosynthetic efficiency will decrease, defective pollination due to pollen sterility, abortion of pod and flower, compromised seed quality, less reproductive structure formation, due to high temperature and more frequent droughts. There are reports of 2 to 4% decrease in yields of soybean for every degree rise in temperature in the  USA between 1994 and 2013 resulting in losses of US$11 billion (Mourtzinis et al., 2015).
➢ Recognition of great threat to the environment during Synthetic production of ammonia by the Haber-Bosch process and its utilisation as N fertilizer, there is more preference on utilizing nitrogen by biological nitrogen  fixation.  As the energy required by Haber-Bosch is about 1.5% of the global total primary energy consumption and  ill effect of nitrogen fertilizers are many like decreasing nitrogen-utilizing efficiency, biodiversity threat due to eutrophication, production of nitrous oxide and other nitrogen by-products of reactive nature. Now there are  many challenges in front of scientists to improve N₂ fixation as it is affected by soil acidity, drought, N fertilization and unavailability of nutrients (Ladha et al., 2016; Tilman et al., 2002).
➢There is large yield gap for legume crops in developing countries like India, Pakistan and Ethiopia17. In Africa, it is more than 300% along with yield of cowpea is only 10 to 20% of their genetic potential. So there is urgent need of improved crop management practices to fill this gap (Anderson et al., 2016).
➢ Some newly discovered perennial legumes with long and deep roots have future potential in providing hydrological stability as they increase access to water low-input agricultural ecosystems. But both conditions; flooding and  drought, pose greatest challenges to the cultivation of soybean and other forage legumes (Striker and Colmer, 2017).
➢ There is urgent requirement to categorise these legume under orphan (neglected one) category to draw the attention  of researchers and industries so that legume cultivation can be increased by improving biotechnical and genetic  traits. Some legumes like grass pea, cowpea, the ‘dolichos’ bean, the marama bean and the tepary bean are already  under orphan category and are being grown in arid regions (Cullis and Kunert, 2017).
➢   There is a scope to find out that, by which mechanism S- deficiency limits SNF and different process of S-metabolism in determinant and indeterminate nodules. Also to find out the compatibility of SOB and N₂-fixing bacteria at  molecular level.

The importance of grain legumes as nutritional, health and environmental point of view is unquestionable, as they constitute many macro and micro nutrients. Even though widespread benefits of legumes, most have neither been taken seriously for their potential production. A coordinated research is needed to explore the enormous genetic variation, N, S and other nutrients requirement and understanding mechanisms at molecular level to improve the biological N₂ fixation. The appropriate focused research on legumes and their acceptance in each and every aspect can allow the legumes in helping towards improving human health, nutritional security and sustainable food practices.

The first author is highly thankful to DST (INSPIRE), Department of Science and Technology, Technology Bhavan, New Mehrauli Road, New Delhi, for providing Inspire fellowship during the entire Ph.D. programme.