Molybdenum as an Essential Element for Crops: An Overview an Essential Element for Crops: An Overview.

Background: Molybdenum for plant growth is essential micronutrient wherever in enzyme catalysis it is required as catalytically active metal. Functional roles are fulfilled by molybdenum in enzyme systems in plants. Enzymes more than 50 Mo-containing are recognized, maximum of them are of bacterial origin, while a few Mo-enzymes are seen amongst eukaryotes. Five Mo-enzymes in plants, to this end are identified: AO, NR, SO, mARC and XDH which catalyze vital significant reactions in degradation of purine, synthesis of phytohormone, nitrogen assimilation and detoxification of sulfite. Scope: The enzymes are significant regardless of having common structural elements, in the series of diverse chemical reactions those are being catalyzed, even though nearly all reactions are two electron oxidation reduction in this an atom of oxygen is transferred from or to the Mo. XDH family enzymes are described best mononuclear enzymes having molybdenum. This article will emphasis on computational approaches to those plant enzymes which requisite Mo as catalytic metal, focusing on their functions, key mechanisms, current prospects and future challenges. Conclusion: Plants distress from deficiency of Mo is limited in growth and development, their leaves shows paleness, disorders in flower formation and ultimately withers. So, molybdenum nutrition for healthy growth and development of plant is key essential where it is being obligatory as a metal that is catalytically active in catalysis


Introduction
Molybdenum in enzymes as catalytic center has chemical adaptability, which is valuable to biological systems, in physiological conditions, it is redox-active. To catalyze diverse redox reactions molybdenum, possess versatile redox chemistry which is utilized by enzymes. At enzyme environment and Mo atom, this redox chemistry is controlled both by different ligands [1]. The molybdate anion in soils is the only Mo form available to plants. For life enzymes having Mo are of vital importance, subsequently they have significant positions both in the redox biogeochemical cycles of N, S and C on earth and in the individual organism metabolism [2][3][4].
More than 50 Mo-containing enzymes are known to be Mo-dependent, Mo five enzymes in plants still are known: sulfite oxidase (SO), mARC (mitochondrial amidoxime reductase), XDH (xanthine dehydrogenase), NR (nitrate reductase) and AO (aldehyde oxidase) Table 1 [5,6]. In cytosol NR is localized that catalyses the nitrate into nitrite reduction. By NiR (nitrite reductase) that is to be found in plastids, into ammonium nitrite is reduced and into amino acids this ammonium via GSGOGAT cycle this is further assimilated. In assimilation pathway of nitrate as NR is the key primary enzyme that is why in plants deficiency of Mo often leads to N deficiency. To peroxisome SO is localized that oxidizes the sulphite (toxic) into sulphate, in opposite way to the assimilation of reductive sulphate that take place in plastids [7]. For purine degradation XDH is required in N-metabolism and can produce ROS (reactive oxygen species) and AO takes part in ABA (abscisic acid) synthesis, glucosinolates, auxin, and probably, further compounds in plants [8][9][10][11].  (Mendel, 2011). Once molybdate come into the cell it is consequently integrated through the complex biosynthetic machineries into metal cofactors. Then these metal cofactors into different enzymes are incorporated [12]. There are two molybdoenzyme distinctive forms, molybdenum nitrogenase have unique cluster molybdenum-iron-sulfur, called FeMoco [13]. The reduction of atmospheric dinitrogen to ammonia is catalyzes by nitrogenase. Other molybdoenzymes are oxidoreductases that transmit two electrons or an oxo group to or from substrate. They possess Mo cofactor in that Mo is coordinated to a dithiolene group on pterin 6-alkyl side chain called MPT (molybdopterin) [14,15]. This review will concentrate on computational approaches to enzymes containing Mo, focusing on their functions, key mechanisms, current prospects and future challenges.

Excess in Crops
Molybdenum insufficiencies predominantly related with poor N health mainly when nitrate is major available N form for These are due to vascular bundles insufficient differentiation on initial leaf development phases and the local necrosis in tissue [16].     Dephosphorylated NR cannot be inhibited by inhibitor protein.

NR and Its Function
Starting at nitrate transporters, signal transduction flow and nitrate linking availability to the transcription induction is still mysterious, but in research receives a lot of attention nowadays. There are signs that nitrate not only serves as substrate for assimilation, but also for coordinating C and N metabolism as regulatory signal and driving root development [54][55][56][57][58].
NR efficient turnover as nitrate reductase as conflicting to nitrite reductase duty depend either on nitrite efficient expulsion from active site, or onto the electronic structure distinctiveness of the active site of Mo that will favor nitrate reduction and binding over nitrite reduction and binding [59]. In current years nitrate reduction has been computationally explored at Mo center of NR, from crystal structure data particularly benefiting [60]. Around active site, significant amino acids were recognized, and their role in fine-tuning reactivity towards the electrons and nitrate were explored in some detail [61]. By NR the nitrite reduction exact mechanism is unknown. This mechanism might include coordination of nitrite via one or two oxygen atoms, or via N.
As another plant NR regulatory region, N terminal extension preceding the Moco domain was shown to be involved in the posttranscriptional regulation by light [62]. NR specific forms NADH in higher plants are most abundant; however specific forms NADPH occur among fungi [63,64]. It is significant that inside protein Moco is completely buried and cone like structure leads from the protein surface to active site. Whereas in nitrogen assimilation nitrite is further reduced to ammonium by nitrite reductase in chloroplasts, by NR itself it can also be reduced to signaling molecule nitric oxide (NO) [65]. As NR post-translational modification also modulated the rates of NO production, it was determined that indeed NR is a active nitrogen species producer also in vivo [66].
Accordingly, grown under Mo deficiency disorders in spinach plants, NR activity in the leaf was found to be decreased and final plant yields grown on sufficient levels of Mo, overall lower than control plants [18,19]. Mo insufficiency in wheat, was also revealed to reduce maximum activities of NR irrespective of the NR regulatory control through periods dark and light [67]. As foliar spray Mo resupplying or in nutrient solution supplemented in maximum instances will recover readily NR activity [19]. In Merlot phenotype present it would indicate not related to activity and synthesis of Moco or NR apoenzyme but associated with a disturbance in mechanism that are monitoring Mo uptake or inner redistribution in phloem and xylem [22].

AO and Its Function
AO enzymes are alike to XDH upon an early gene duplication that is derived from XDH. Coextensive with this, both enzymes show a high degree use of same prosthetic groups and sequence similarity which catalyze the oxidation of a variety of nonaromatic and aromatic aldehydes and heterocycles, so converting them to respective carboxylic acid as they share a more degree of sequence homology, so that during evolution it is presumed that from XDH, AO has been derived by neo-functionalization and gene duplication.
Then XDH, AOs display a greatly substrate specificity, covering aldehydes, aliphatic and aromatic heterocycles as well as pteridines and purines while 300 kDa apparent molecular mass and therefore with XDH shares structural and catalytical similarities. AO proteins in contrast to XDH preferably oxidize aldehydes to respective carboxylic acid. Furthermore, during catalysis molecular oxygen is exclusive electron acceptor and its consumption is obligatorily linked with the generation of hydrogen superoxide and peroxide anions [9,68]. However, main differences are present regarding the binding of substrate at center of Mo and physiological electron acceptor [69,70]. AO is a severe oxidase that is being not capable to bind NAD but solely with molecular oxygen usage as electron acceptor. The fact that IAA belongs to the plant hormones auxin family proposes a probable physiological role of AO enzymes in biosynthesis of auxin throughout plant early stages development.
The characteristics most prominent that differentiate AO from XDH enzymes found to concern binding of substrate at the molybdenum center and physiological electron acceptor binding [71].
In A. thaliana four AAO1-AAO4, AO genes were recognized whose products forms the heterodimers, homodimers as well as thus lead to changed respective isoenzymes substrate specificities [72][73][74]. For AAO1 and AAO2, both efficiently catalyze in vitro indole acetaldehyde oxidation to indole-3-acetic acid. AAO4 in siliques is expressed preferably and catalyzes the benzaldehyde oxidation into benzoic acid and latter being amalgamated into glucosinolates that serve as defense compounds of herbivore [11]. AO enzymes are the firm oxidases that unable to bind NAD+ and solely usage molecular oxygen as an electron acceptor, thus generating hydrogen peroxide [9].AAO1 and AAO2 gene products, in seedlings six day old form heterodimers and homodimers, AO isoenzymes that are capable of making indole 3 acetic acid [72]. Arabidopsis mutants with AAO3 deficiency hence by reduced level of ABA are characterized accompanied through excessive loss of water and wilty phenotype, reduced stress tolerance and stunted vegetative growth [74].
AO isoform AAO3 by abscisic aldehyde turns superlative as substrate. Abscisic aldehyde is native precursor of absicisic acid plant hormone that is vital for numerous growing processes as well as for a variety of biotic and abiotic stress responses [75][76][77].
Another AO isoform known is AOδ that is required for abscisic aldehyde oxidation producing the abscisic acid phytohormone [8]. For various developmental processes and for various biotic and abiotic stress responses, Abscisic acid is essential for these [77]. Mutant analysis and tissue distribution from the specificity of substrate it could be concluded that Arabidopsis AO3 catalyses the transformation of abscisic aldehyde to ABA (abscisic acid), the final step in ABA-biosynthesis [78,79]. In the IAA over producing mutant sur1, AO1 activity is being found five times greater as compared to wild type of Arabidopsis [80]. Thus, for numerous physiological processes, AO is extremely significant in plants including the onset of senescence. Other AO isoenzymes are involved in auxin phytohormones biosynthesis in early plant development stages.
By high preference, the homodime AAO3 is considered for the abscisic aldehyde as substrate [74], that is the ultimate ABA precursor which is involved in numerous features of plant development and growth, together with maturation of seed, leaf senescence dormancy, and adaptation to diversity of environmental stresses absolutely use the molecular oxygen as electron acceptor. Upon transfer of substrate-derived electrons to molecular oxygen, plant AO generates hydrogen peroxide [9]. Recently, levels of AAO3 protein by ubiquitin reliant degradation have shown to be controlled via 26S proteasome to avoid premature senescence through ABA accumulation [81]. This proposes that during the senescence onset, in AO proteins the synthesis of ABA also play an acute role that needs a tight control of levels of AO and ABA.
From maize, Arabidopsis and tomato, the AO gene has been cloned where four AO cDNAs were seen and mapped physically to dissimilar chromosomes [82,83]. The isoforms of encoded enzyme plants deficient in AAO3 are described by more transpiration rate, impaired seed dormancy and reduced stress tolerance [84].

XDH and Its Function
XDH is the important key enzyme of purine degradation. XDH needs FAD, Moco, and the two iron sulfur clusters [85]. XDH like all Mo-enzymes, is a functional dimer comprises of two alike subunits.
By a molecular mass 300 kDa plant XDH is homodimeric [86] and XDH was seen also to catalyze ROS substrate independent formation because of an intrinsic NADH oxidase activity. XDH is active as a homodimer of two alike subunits, each one being subdivided in distinct three domains, N terminal domain for binding two clusters activities. At NAD+ tremendously lower concentrations, as alternate electron acceptor molecular oxygen can assist with simultaneous superoxides generation [88]. Plant XDH production of ROS may be of physiological significance because XDH increasing activities and ROS simultaneous production were detected upon interactions of plant-pathogen, natural senescence, drought stress, hypersensitive response and virus infection [89][90][91].
During senescence oxidative processes comprise enzyme activities increase producing superoxide ions and oxygen radicals.
In leaves of pea, activity of XDH was increased sharply in analogous with enzymes related to oxygen and superoxide dismutase [92].
The XDH function is crucial as showed by plants that are deficient in XDH, for plant growth, fertility and senescence [93]. XDH displays strong intrinsic activity of NADH oxidase that is complemented by consumption of oxygen as an electron acceptor and simultaneous superoxide anions formation [88]. It is guessed that this key activity has significance in the abiotic and biotic stresses response. With microbodies XDH could be related and later this was described that peroxisomes in pea leaf have activity of XDH that catabolizes inside the organelles, xanthine to uric acid. In contrast, in nodules of cowpea, XDH immunocytochemistry revealed a cytoplasmic location, and in the Arabidopsis XDH sequence no targeting signal was found [94,95]. In Arabidopsis two genes were founded that are situated in genome side by side, encoding XDH1 and XDH2 isoenyzmes [90]. Activity of XDH increases when the phytopathogenic fungi Uromyces or Puccinia infect legumes and cereals [96]. Whether this response is aimed at oxidative defence mechanisms it still unknown; however, in pea, XDH activity is strongly correlated with the activity of superoxide dismutase [92].
The fixed nitrogen export and mobilization out of nodule needs the molybdoenzyme XDH activity. Dependent on species of legume, fixed N is exported as either ureides or amides, that are construction of ROS also with superoxide simultaneous production, activities of NADH oxidase have been revealed for plant XDH [88].
As this activity is highly prominent, this suggests that XDH is an effective superoxide producer in vivo also, and that enzyme could be involved in NADH and NAD cellular regulation balance.

SO and Its Function
The SO enzyme is significant for sulfite detoxification, i.e. the sulfite oxidation to sulfate and has 90 kDa molecular mass for dimer. In chloroplasts in the course of primary sulphate assimilation, sulphate via sulphite is reduced to organic sulphide that is used for biosynthesis of cysteine [97][98][99]. In plant kingdom the gene SO is greatly conserved and widespread. As redox center, SO retains only Moco and together with mARC, is the simplest Mo enzyme found in eukaryotes. However, Plant SO enzyme, is peroxisomal enzyme that as electron acceptor uses molecular oxygen and during catalysis forms hydrogen peroxide simultaneously [7,100,101]. The latter fact could describe the SO peroxisomal localization since during oxidation of sulfite, excess hydrogen peroxide generated, by catalase may eliminated easily. As sulfite is being sturdy nucleophile that can react with a wide range of cellular components it was supposed that SO possesses function of sulfite detoxifying and is necessary for eliminating surplus sulfite from cell [7,27]. In support of this, [27] found independently that in comparison to plants wild type plants deficient in SO are more susceptible to sulfite high concentrations while overexpressing SO plants are more excess sulfite tolerant.

However, in normal situations, in plants the loss of the activity of SO
is not associated to an apparent phenotype, suggesting that rather than housekeeping metabolic enzyme, SO symbolizes salvage enzyme [102]. Though, it also been described that back to sulphate, sulphite could be oxidized, e.g., when SO2 gas subjected to plants or, when the isolated chloroplasts were fed with labelled sulphite radioactively. Activity of Sulphite oxidizing was identified in the dark and light [103,104]. Therefore, sulphate assimilation would be counteracting by SO, providing that in chloroplast it would be localized. In normal conditions, SO is submitted to be sulfate sulfite cycle part which is highly important for sulfur distribution fine tuning in cell.
The compartmentalization of sulphur assimilation and sulphite oxidation in different organelles allows plants to coregulate these opposing metabolic demands. In chloroplasts oxidation of sulfite was seen to be increaseable by the light and sensitive to photosynthetic electron transport inhibitors and thus due to the reactions that are non-enzymatic was interpreted to be mainly during the electron transport [105]. Though, there exists a new sulfite oxidizing activity that also happens in dark and can be pelleted when isolated chloroplasts are broken. From spinach chloroplasts, latter activity was purified, and it has been found to be connected with the thylakoid membranes [103,105,106]. Plant SO known to date amongst eukaryotes, the one that is lacking active redox centres other than the Moco. The association of SOs Moco domains from different sources with Arabidopsis NR and SO shows significant whole homology, pinpointing these enzymes such as members of the common family [98]. Amongst higher plants Plant

SO is conserved as evinced by fact that upraised antibodies against
Arabidopsis SO identified a dominantly protein band cross reacting of almost 45 kDa in an extensive variety of species belonging to range of both woody herbacious plants.

Mitochondrial Amidoximereductase and Its Function
mARC (mitochondrial amidoxime reducing component) was exposed as fifth eukaryotic Mo cofactor having enzyme [107]. For

Mo-enzymes and Moco biosynthesis, all genomes of eukaryotes
well-known to encode the proteins, as well encode two proteins mARC, proposing that the mARC proteins custom an own minor protein family [108]. All mARC eukaryotic proteins comprising the counterparts of the plant, which forecast these proteins mitochondrial localization, are described by N-terminal extensions presence.  In contrast, entirely other Mo enzymes, mARC eukaryotic proteins not show on their own the enzymatic activity but need other proteins alike NADH, cytochrome b5 reductase and cytochrome b5 as electron donors and electron transmitters, respectively thus forming the mARC enzyme complex [110].The two other proteins, CY B5 R (cytochrome b5 reductase) and CY B5 (cytochrome b5 ), from NADH to terminal oxidoreductase, mARC catalyze transfer of electron [110]. NO (nitric oxide) from nitrite can be generated by mARC when forming a chain of electron transfer with NADH, NA-DH-dependent cytochrome b5 reductase and cytochrome b5 . When pH decreased from 7.5 to 6.5, the NO formation rate rises three-fold. [111] conducted the experiment to determine if reduction of nitrite is catalyzed through Mo in mARC-1 active site, they mutated and NADH-CY B5 R [112]. As a sole prosthetic group mARC protein bind Moco [110]. mARC proteins belong to the SO-family most likely, but final confirmation needs to be provided [ Figure 3].

Role of Molybdenum in Abiotic Stress Mediation
Mo have significant roles in resisting numerous environmental stresses, such as drought, salt stress and cold [113][114][115][116][117][118]. Amongst the abiotic stresses low temperature is the most important affecting plant growth, development, distribution and production all over the world. Under cold stress in the winter wheat, Mo was having progressive impacts on drought stress, rates and products of photosynthesis. Through the antioxidative enzymes escalating activities Mo also boosted the turf grass chilling resistance [20, [118][119][120] revealed that Mo reduced Cd concentration under the Cd stress. Moreover, as a result of Mo excess or deficiency it has been described that the biomass, product quality and seed yield all deteriorated [121]. Low temperatures can be tolerated by some plants in a process that is called as cold acclimation, this happens when plants are wide-open to non-freezing low temperature (4 0 C) [122,123]. Throughout the process of acclimation, various alterations happen such as osmoprotectants accumulation alike amines, soluble sugar, and compatible solutes such as proline, polyols, and betaine, through activation of low temperature signal pathways of transduction that ultimately lead to transformed gene expression and membrane stability to provide tolerance at all levels [124][125][126].
In Arabidopsis intensive studies on cold response gene expression led to the DREB1/CBF transcription factors identification that has a crucial role in plants during freezing and cold acclimation stress tolerance [127]. In the cold promoters and genes responsive for dehydration, these transcription factors also bind to sequences of specific regulatory. These sequences are elements responsive for dehydration and C-repeat. These both sequences have the highly 5-bp conserved sequence of CCGAC that has the ability to transcription regulation in drought, salinity and temperature [128]. Therefore, CBF induces the COR genes expression and in plant cold tolerance improvement these genes have an important crucial role. [129,130] reported the Mo optimistic influence on the improvement of cold tolerance in the cauliflower without acclimation.
Earlier study showed that under cold stress, deficiency of Mo inhibits the chlorophyll biosynthesis and leads to chlorophyll decrease in cultivars of winter wheat [131] and through Mo application showed the positive effect on stomatal conductance, photosynthetic rate and decreased transpiration rate in winter wheat under stress of low temperature [118,132].On the other hand, in chickpea excess Mo reduced the biomass, lessened the produce quality and yield of seed yield [121,115] and [133]have stated that for wheat gene expression may be altered not just by decreased in temperature (4 0 C), but subsequent disclosure to certain chemicals that were used for priming of seed e.g. Mo, that can upsurge the expression of CBF. Moreover, this study also indicated that to tolerate decrease in temperature these chemicals improved the ability of plants due to the ABA biosynthesis regulation via AO, and ABA is takes part in mediating of COR (Cold regulated) expression of genes. In the meantime, [115] have described that the activity of AO, IAA and ABA content increased in wheat leaves that were treated with Mo. It also been earlier suggested that in frost damage amelioration, Mo can be involved [133,134].
Worldwide drought stress as a main environmental stress is well known that limits the crop plants development, yield and growth, and can generate a series of biochemical and physiological plants responses [135]. The main biochemical and physiological characteristics not just associated to photosynthesis inhibition, decline in transpiration, decline in chlorophyll content and stomata closure but are involved also in ability of osmotic adjustment and antioxidant that are significant ways to increase resistance of plant to drought capacity [136]. Numerous studies have presented that drought stress may result in more active oxygen species production such as hydroxyl radicals, superoxides and superoxide radical [137], that results membrane damage and cell death [138][139][140] reported that the antioxidant enzymes activities such as superoxide dismutase, ascorbate peroxidase, peroxidase, catalase, and nonenzymatic antioxidants content like ascorbic acid, carotenoid, (ascorbate peroxidase) and POD (peroxidase) or through enhancing non-enzymatic antioxidants levels such as, GSH (glutathione), AsA (ascorbic acid) and CAR (carotenoid) [141,142].
Furthermore, in plants to resist stress of drought osmotic adjustment also is significant way. The substances that help in osmoregulation such as proline, soluble proteins and soluble sugar also show important roles in osmotic equilibrium maintenance and under deficiency water the integrity of membranes [143].
The wheat drought tolerance might be improved through the application of Mo by enhancing utilization capability of water, antioxidative defense abilities and osmotic adjustment. It is described also that overexpression sulfurase gene Mo cofactor confers drought tolerance in tobacco, soybean, cotton and maize [144]. Environmental adversative conditions comprising soil salinity, heavy metal contamination and water stress can affect intensely on plants nitrogen assimilation, this rises from the stress effect on nitrogenase and nitrate reductase enzymes activity. [116] have showed that Chinese cabbage fresh weight increased significantly by the Mo application under the salt stress. They also indicated that under salt stress the nonenzymatic antioxidants contents and antioxidant enzyme activities were extraordinarily enhanced by application of Mo. By Mo application accumulating products of the Osmotic adjustment were also increased dramatically under salt stress. Furthermore, the ratios Na+-K+ were increased intensely and with the Mo application rates were correlated positively. Hence, we can see that the Mo application tolerance to salinity was improved by the osmotic stress tolerance and increasing eliminating active oxygen ability. [145] studied the Mo deficiency stress effect on viability, seedling growth and seed germination of wheat and described that deficiency of Mo results in necrotic spots on wheat leaves and wrinkled and poor seeds. [67] also reported a remarkable reduction in activity of nitrate reductase and metabolism of nitrogen under deficiency of Mo [146][147][148][149][150].   53. Campbell WH (1999) Nitrate reductase structure, function, and regulation. Bridging the gap between biochemistry and physiology. In