Cadaverine

Abstract

The cadaverine (Cad) a diamine, imino compound produced as a lysine catabolite is also implicated in growth and development of plants depending on environmental condition. This lysine catabolism is catalyzed by lysine decarboxylase, which is developmentally regulated. However, the limited role of Cad in plants is reported, this review is tempted to focus the metabolism and its regulation, transport and responses, interaction and cross talks in higher plants. The Cad varied presence in plant parts/products suggests it as a potential candidate for taxonomic marker as well as for commercial exploitation along with growth and development.

Introduction

Lysine an essential amino acid for mammalians is supplemented through daily dietary intake. The efforts to increase the lysine by genetic manipulation to elevate its concentration in the plant parts to mitigate the nutritional deficiency in starving population are consistently being made.¹ The lysine catabolites are implicated in various processes in plant’s life cycle. In osmotic stressed plant, lysine catabolised in acetyl CoA along with intermediates glutamate, pipecolic acid by the enzymes lysine keto-glutarate reductase (LKR) and saccharopine dehydrogenase (SDS).² The amine adipate another catabolite of this pathway is implicated in osmoprotection in bacteria as well as in plants.³ However, Galili et al.⁴ suggested that under stress and under some other conditions, lysine change into glutamate by some novel mechanism, which is considered to be developmentally regulated and that might be important for mammalian brain system. This may be likely in plant also. In fact, the regulation of lysine synthesis and catabolism is not elucidated convincingly except that balancing of lysine concentration as demonstrated in seed.⁵ However, the high lysine level in transgenic of bacterial Dihydrodipicolinate synthase (DHPS) in Arabidopsis either it’s overexpression or knockout LKR/SDH mutant⁶ led to inhibition in seed germination. The lysine metabolism involvement in seed germination and growth is further confirmed. The knockout and co-expression of genes LKR/SDH along with bacterial DHPS under seed specific promoter suggested balancing of lysine level and accordingly play role in seed germination or vegetative growth.⁷ This led to proposal that lysine catabolism after germination only inhibits seedling development. Having this considered catabolism, under either plant vegetative or reproductive stages of plants, a hypothesis is forwarded that this is an essential component for plant growth, but depends on environmental conditions.² The polyamines (PA’s) including diamines are implicating in growth and development of plant. Another diamine, putrescine (Put) is a precursor of polyamines spermidine and spermine, synthesized in the presence of enzyme spermidine and spermine synthase respectively. These aliphatic amines are also considered as a plant regulator and widely noticed in plant at different developmental stages. It is envisaged that polyamines/diamines may act as a nutrient for protozoans (Trypanosoma cruzi), an animal parasite and the Put is being transported to those animals as an obligatory precursor for many metabolites.⁸

Moreover, in addition to above mentioned pathways of lysine catabolism, it’s decarboxylation by an enzyme called lysine decarboxylase (LDC) results in to damine called cadaverine (Cad) i.e., 1,5-diaminopentane, demonstrated in many organisms including plants. Since the LDC (EC 4.1.1.18) presence marked in some bacteria, cyanobacteria⁹ and in higher plants particularly in gramineae, leguminoseae and solanaceae,^10-12 indicates the Cad wide and varied presence.

Recently, Ohe et al.¹³ suggested that the ldc genes are varied in number and sequences differentially expressed depending on the plant organ, plant growth stage and environmental conditions, for instance 3 in Arabidopsis and 9 in rice. The ldc gene having only 1 conserved domain in rice and Arabidopsis as compared 3in bacteria after domain analysis through Pfam database (Fig. 1).

Figure 1. LDC protein domain analysis using Pfam database (http://pfam.sanger.ac.uk/) in Bacteria (A), Arabidopsis and Rice (B).

However, Brieger¹⁴ suggested that Cad and putrescine, both diamines are bacterial decomposition product, which is smelly and volatile in nature. The foul odour of putrefying flesh of cadaver is due to cadaverine¹⁵ and so its name. In view of above, the present review is focused to look into Cad presence and functions in higher plants, so as to throw some light on future prospects.

Cadaverine Occurrence and Distribution

In whole plant and plant product

The Cad presence was initially marked in bacteria in very low amount and attributed in many functions due to its binding potential to membrane and other molecules.¹⁶ This diamine has been reported from many plants, their parts and also from products (Table 1). It is reported in rice, millet (Panicum miliaceum and Setaria italic), oat, rye, wheat, barley, maize, sorghum and Phleum pretense^10,11,17,18 except broad bean cotyledons and axial parts¹⁹ and cucumber fruit.²⁰ The tissues Cad presence is reported from leaf of barley,²¹ root of pea, tomato²² and micro cutting of grape vine,²³ tea plants.²⁴ The vegetables like Chinese cabbage²⁵ iceberg lettuce and radicchio²⁶ also contain Cad. Broquedis et al.²⁷ have demonstrated that Cad is either present in traces (in leaf and bunches) or absent in Vitis vinefera. It is also deficient in Pisolithus tinctorius.²⁸ It is suggested that Cad mainly localized in the radicle and hypocotyl region of chick pea,^29,30 whereas absent in nodules of soybean³¹ as well in ripening egg plants.³² Fujihara et al.³³ reported a new polyamine, 4-amino butyric Cad in root nodules of adzuke bean. In addition to presence of Cad in various plants, it is reported in table olive,³⁴ sugarcane juice.^11,35 It is challenging to define the relevance of Cad in these products. The variability in content is a less understood and remain unelucidated in terms of physiochemical significance.

Table 1. Cadaverine occurrence and distribution in plant species and their parts.

Plant species	Plant Part	References
Adenocarpus decorticans	Seed	38
Adzuke bean	Root nodules	33
Agaricus bisporus	Fruiting body	54
Allium sativum	Leaf and shoot	53
Astragalus granatensis	Seed	38
Avena Sativa	Leaf	18
Boletus badius	Fruiting body	54
Boletus chrysontereon	Fruiting body	54
Boletus variegatus	Fruiting body	54
Brassica napus	Leaf	89
Brugmansia candida	Root	41
Brugmansia candida	Hairy root	41
Dianthus caryophyllus	Leaf	71
Cicer arietinum	Seed	49
Brassica Pekinensis	Upper ground part	25
Chinese cabbage endive	Leaf	26
Cicer arietinum	Embryonic axes	142
Citrus species	Immature seeds	55
Cucumis sativus	Cotyledon	65
Cytisus reverchonii	Seed	38
Eichhornia crassipes	Root	18
Festuca indigesta	Seed	194
Glycine max	Mature cotyledon	82
Hordeum vulgare	Leaf	21
Lactuca sativa	Leaf	26
Lupinus angustifolius	Seed	49
Lycopersicon esculentum	Root	89
Lycopersicon esculentum	Bud and root	67
Mesembryanthemum crystallinum	Leaf, root and stem	57
Nelumbo nucifera	Leaf	18
Nicotiana glauca	Leaf, Root and stem	10
Nicotiana tobacum	Hairy root cultures	68
Nymphea tetragons	Leaf and root	18
Olea europaea	Table olive	34
Oryza species	Phloem sap and shoot	52
Panicum miliaceum	Leaf	18
Phleum pretense	Leaf	18
Pisalithus tinctorius	Mycelium	147
Pisolithus tinctorius	leaf and bunches	28
Pisum sativum	Inter node	50
Pisum sativum	Root	22
Radiocchio	Leaf	26
Saccharum officinarum	Juice	195
Sorghum bicolor	Leaf	18
Trifolium subterraneum	Seedling	17
Vicia faba	Seed	49
Vitis vinifera	Leaf	48
Zea mays	Coleoptiles	22

The Cad titer may vary from < 1 nmol/g FW to 15 mol/g FW depending on cereal species and parts.³⁶ Tobacco hairy roots accumulated Cad 0.7% on dry mass basis, suggesting involved in anabasin synthesis.³⁷ Though, Cad presence was marked in Chinese cabbage but Put considered to be dominating.²⁵ Angosto and Matilla,³⁸ mentioned that 3 leguminous species (Adenocarpus decorticans. Astragalus granatensis ssp. granatensis and Cytisus reverchonii) that had variation in diamines titers may depend on altitude as high altitudinal plants had more Cad.³⁹ The temporal changes are also noticed in Cad level in different plants. Cad declined with increased age in pea, tomato, millet and corn internode as well as corn coleoptiles^22,40 and grapevine leaves.²⁷ Carrizo et al.⁴¹ have also demonstrated age dependent Cad titer in Brugmansia candida and concluded that it may decline progressively in hairy roots.

Seeds of broad bean, chickpea, cucumbers and lupin contain high amount of Cad, which increases during germination.^42-49 In pea seedlings, 3rd internode also contain higher amount of Cad.⁵⁰ It corroborates the result of Villanueva et al.⁴⁵ but contradictory to that of Smith and Wilshire,¹⁷ who could not detect Cad in Phaseolus aureus, P.vulgaris and Vicia faba except in Trifolium subterraneum seedling. Though, thereafter studies reported Cad from hypocotyls and roots of soybean²⁹ and confirmed its presence in other legumes.⁵¹ Yokota et al.⁵² demonstrated Cad presence in phloem sap and shoot exudates of rice and suggested wider distribution from bacteria to plants to mammals.

Besides crop plants, Cad is also reported in leaf and root of some aquatic plants like Nymphea tetragons (water lilly), Nelumbo nucifera (lotus),¹⁷Spirodela polyrhiza (duckweed) and Eichhornia crassipes (water hyacinth).¹⁸ Good amount of Cad in leaf and shoot of Allium sativum, but highest amount (165 mg/ dry wt.) in edible mushroom Boletus badius, B.chrysontereon, B.variegatus and Agaricus bisporus have been observed.⁵³ However, Cad content is found lower than that of Put (600 mg/dry wt.) in plant system.⁵⁴ Han et al.⁵⁵ recorded high amount of Cad in immature seeds of citrus species and in straw mushroom⁵⁶ as well. The Cad presence is noticed in brines of green table olives.⁴⁸ The ice plant, young leaves and apex had lower level of Cad (20–29 n mol/g Fwt) than the root and stem.⁵⁷ The ice plant Cad in old leaves, stem and roots appeared to be heat labile, i.e., 47°C for 2h reduced the level drastically.⁵⁸

Krizek et al.³⁹ examined Cad in 53 grasses and 54 maize silages and reported Cad in range of 642 mg/kg and 341 mg/kg respectively. However, higher Cad content in grass silage is confirmed.^59,60 The physical factors affecting the Cad titer might be modulating at either lysine, the precursor or LDC enzyme protein or its expression. Goren et al.⁵⁰ recorded Cad variations in pea subjected with red and far red light. Cad massive accumulation is observed in rape leaf disc subjected to osmotic and drought stress.⁵⁰

Hou and Lin,⁶¹ also have recorded different titer of Cad in storage roots, sprouted roots and sprouts of Ipomoea batatas and a similar trend in mungbean, lentil and radish sprouts. It seems that differential distribution in germinating/growing tissue might be due to LDC isoforms, as proved by applying inhibitor α-difluoromethyl l-lysine.¹³ The uptake and distribution of polyamines were influenced by charges.^62,63 The Cad in cereals has been further confirmed by localizing related gene on chromosome 5B fragments responsible for synthesis in wheat.⁶⁴

In Cultured Tissues

The culture of cucumber cotyledon showed higher amount of Cad.⁶⁵ Carrizo et al.⁶⁶ have reported that Cad was only present in transformed root culture of Brugmansia candida. The explants bud and root differentiated callus of tomato also showed increased Cad content than non-differentiating callus.⁶⁷ Fecker and group⁶⁸ noted the increased production of Cad in hairy root cultures of Nicotiana tobacum. The root culture of tobacco contained Cad is being metabolized in secondary metabolites like hydoxy cinnamoyl cadaverine.⁶⁹ Barley contained Cad and aminopropyl Cad APC as well as N, N bis 3-amino propyl Cad.⁷⁰ The micropropagated carnation plants also exhibit high level of Cad conjugates.⁷¹ The variation in Cad titer in cultured cells tissues suggests its role in, growth differentiation /organogenesis.

Detection of Cadaverine

The Cad is determined through the methods applied for all other polyamines. The experiments performed for detection of Cad and other PA in root nodules of pea, fava bean and stem nodule of Sesbania rostrata revealed that 15 N values in Cad was +1 and +7 per thousand, almost similar with detection limit with automatic nitrogen and carbon analysis mass spectrometer. Though, a quick method of HPLC reverse phase is also suggested after preparing benzoyl amine with benzoyl chloride derivative. But this has been detection limit 5.60 to 54.40 pg by using uv-visible spectrometer at 225 and 254 nM.⁴⁸

Cad was estimated by using derivatives of dansyl chlorides with HPLC in rice (Oryza sativa L.) cv El Paso. Whereas lysine decarboxylase (LDC) activity can be assayed by ¹⁴CO₂ production from (¹⁴C)-lysine.⁷² Probably precise detection methods are costly may be the reason that wide range of plants are still remains unexamined for the LDC and Cad.

Cadaverine Biosynthesis

Cad has independent single exclusive biosynthetic pathway (Fig. 2A and B) in all organisms. This is unlike to putrescine synthesized differently in different organism like in animals, fungi and plants from ornithine by ODC and in plants from agmatine by ADC.⁷³ It is also proposed that combined enzymes of intermediates like agmatine immunohydrolase and N-Carbamoyl putescine amidohydrolase also produce Put.⁷⁴ The synthesis of Cad from lysine is also proposed as by product of methionine synthesis through aspartate pathway.⁵⁸ However the lysine decarboxylation by LDC is taking place through pyridoxyal phosphate⁷⁵ but there are few exceptions. Like under ornithine deficiency, ODC can use lysine as an alternate substrate for cadaverine synthesis.⁷⁶

Figure 2.

(A) Pictorial presentation of Ldc C and Cad A genes for Cad synthesis in Bacteria [118](B) Cadaverine biosynthesis and catabolism in plants; LDC = Lysine decarboxylase, DAO = Diamine oxidase. Dotted arrow indicates probabilty of synthesis in some cases and (//) show inhibitory response.

The labeled lysine incorporation in Cad in chickpea seeds, and seedlings confirmed the lysine as a precursor.³⁰ The synthesis of Cad directly regulated by LDC, localized in chloroplast stroma,⁷⁷ is demonstrated through accumulated Cad dependent alkaloid quinozolidine level.⁷⁸ Ohe et al.¹³ have demonstrated correlation between Cad level and LDC activity in germinating Glycine max and observed peak on second day of germination. However, radiolabelled 15N-lysine and 15N- cadaverine experiments indicated differential synthesis and translocation in tissues under dark/light conditions. This study suggested least Cad synthesis in cotyledons of Glycine max.

The ¹⁴C lysine experiments also revealed the LDC activity and formation of labeled Cad and ¹⁴CO₂ evolutions in embryo axis, cotyledons, epicotyle, hypocotyle in seeds and roots of C. arietinum resulted.⁷⁹ Based on decarboxylation rate in soluble fraction of enzyme preparation suggested the presence of isoforms in different organs of seedlings. Recently, Bansupa et al.⁸⁰ demonstrated the presence of phyto-genetically distinct subfamily of LDC based on cDNA transcript screening from different group of plants. However, the recombinant LDC and ODC (from L. angustifolius) experiments approves the chloroplast localization and transient expression in A. thaliana and both lysine and ornithine as substrate, where lysine was preferred indicated through site directed mutagenesis.

The LDC using lysine and ornithine as a substrate could be inhibited by DFML irreversibly but induced with lysine at low pH. Infact, lysine being constitutive precursor for Cad in S. ruminantium was completely blocked by DFML and DFMO.⁸⁰ This seems to intrigues control of Cad synthesis in different type of organism. The Mycoplasma dispar also exhibits LDC inhibition by DFML and DFMO.⁸¹ The absence of any other 15N-derivatised polyamines during supplementation of exogenous 15N-lysine in to 15Cad is suggestive of some indirect effect on the other diamines or tri or tetra of the group. Which was further substantiated by 15N Cad.¹³ However, in legumes the biosynthesis is proposed to be ontogenic and suggested to be higher only during growth period of embryonic axes than that of the developing and mature cotyledon of G.max.⁸² The downregulation of Cad in mature seed embryo due to defoliation and shading is also proposed.⁸² The reduction of ldc gene expression in rice with different developmental stages (Fig. 3) using gene investigator software is predicted (www.geneinvestigator.com). The biosynthesis may be affected by other factors also. Ethylene treated pea seedlings reduced the ADC activity but increased acitivity of LDC thereupon increased the content of cadaverine and in M. crystallinum under salinity stress.^58,83

Figure 3. Pictorial presentation of LDC gene (LOC_Os05 g46360) expression in rice at different developmental stages using gene investigator software (www.geneinvestigator.com).

Cadaverine Catabolism

Amine of oxidases are major catabolising enzymes of polyamines. The cadaverine oxidation is primarily through diamine oxidase (EC 1.4.3.6) in apoplast.⁸⁴ This leads to production of 5-aminobutanal, ammonia and H₂O₂.⁸⁵ The Arabidopsis genome mapping revealed total 12 diamine oxidases genes out of which ATAO1 is characterized.⁸⁶ During seed germination the diamine oxidase activity seems to be developmentally regulated which was peaked consistently at 5th day.⁸⁶ Apparently there appears to be link between inhibitors of Cad synthesis and diamine oxidases. Latter was found increasing in case of DFMO.⁸⁷ It is surprising that DFMO action appears to be tissue and compound specific because the DFMO inhibited the LDC activity so as Cad but not the synthesis of Putrescine and others in meristematic tissues.⁸⁷ This might be due to arginine synthesis pathway operativity. The suggestion of nonspecific inhibition of LDC by DFMO further shows crosstalk between diamine and polyamines and possibility of allosteric regulation. This is worth to explore and the information may be relevant in molecular modulation of the diamines, thereby changing nutritional quality or pathogenesity. DFMA (DL-difluoromethyl arginine) a potent inhibitor of ADC decreased the Cad content in stressed leaf suggests non-specific inhibition of the Cad synthesis.⁸⁸ It is found that DFMA being inhibitor of Put synthesis reduced Cad level up to 95%, while DFMO (DL-difluoromethylornithine), another inhibitor of Put synthesis from ornithine, did not alter the level of Cad in rape leaf disc especially under stress.⁸⁹ This non-specific effect on Cad synthesis indicates indirect regulation of polyamines contents depending on plant species and physical condition. This indicates that Cad biosynthesis might be influenced by general metabolism of PAs. This crosstalk needs to be elucidated, especially when plant containing all the precursors of polyamines such as arginine, ornithine and lysine, but Cad presence remains unreported from wide range of higher plants. There is lack of clear correlation between Cad biosynthesis and Put/Spd/Spm. It could be forwarded that plant might be enabling to compensate its PA’s balance, as proposed in animal system,¹³ that need to be examined further for plants.

Generally, polyamines are largely metabolized into alkaloids; few are responsible for specific alkaloids synthesis.⁹⁰ Cad is also regarded as precursor for the biosynthesis of quinolizidine, piperidine and other types of alkaloids.^91,92 Apart, the Cad conjugation in Mesembryanthemum crystallinum root induces oxidative burst concomitantly declining in Cad and its conjugates under exogenous Cad treatment of the root.⁹³ This suggests some wider involvement of Cad metabolites. The Cad conjugate like 4-amino butyric Cad is synthesized in root nodules of Vigna angularis.³³

The Cad linked alkaloid synthesis, catalyzed by N-methyl transferase in tobacco.⁹⁰ However; Cad (K_i 0.04mM) inhibited the Put-N-methyl transferase (PMT), the first enzyme of tropane and pyrrolidine alkaloids synthesis.⁹⁴ However, the LDC activity is positively correlated with the accumulation of quinolizine alkaloids derived from Cad, as a precursor.⁷⁸ Wink and coworkers⁹⁵ have suggested that the decarboxylation of lysine to Cad is the first step of quinolizidine alkaloids biosynthesis. The transgenic Nicotiana glauca hairy root cultures containing the gene coding for a bacterial LDC showed enhanced levels of lysine decarboxylase, resulting into Cad and derived in to anabasine alkaloid and hydroxylcinnamoyl.^68,69 Cad is considered as precursor for piperidine alkaloids in Nicotiana and the quinolizidine alkaloids in Lupinus.⁹⁶ Cadaverine pyruvate transaminase is principal step in quinolizidine alkaloid biosynthesis in Lupinus cell culturing,⁹⁶ but not in the synthesis of tropane alkaloid in case of datura, whereas in tobacco, almost all alkaloid synthesis is induced after feeding Cad to cotyledons.⁶⁴

The growth regulators conjugate formation or derivation in to other form is a natural homeostasis. So, alkaloid derivation might be helping in maintaining homeostasis of polyamines and/or Cad in plant tissues for its specific requirement. This is understandable with the observation that Cad inhibited (K_i 58 µM) homospermidine synthase, a first enzyme of alkaloid specific pathway, which catalyzes the synthesis of homospermine by use of 2 molecules of Put in presence of NAD⁺ in root of Senecia vulgaris.⁹⁷ Though, both diamine Cad and Put, and tetramine spermine did not show any effect on berberin synthesis in Thalictrum minus cell suspension culture, while spermidine induced synthesis of berberine, may be possibly via ethylene generation.⁹⁸ It indicates that Cad is not involved in ethylene production, suggesting its proximity in functional variation like other members of the group. However, interestingly putrescine hydroxyl cinnamoyl transferase, though specific for Put dependent alkaloids, can also use Cad as a substrate.⁹⁹

Siebela et al.¹⁰⁰ suggest that Cad hydroxylation either giving 2-hydroxy Cad or 3 hydroxy Cad can be further oxidized by pea amine oxidase into either 5-amino-4 hydroxy valeraldehyde or 5-amino-3-hydroxy valeraldehyde, further metabolised by aminoaldehyde dehydrogenase producing H₂O₂ and NH₄. Jackson et al.⁷⁰ demonstrated Cad and its higher homologous amino propyl or N, N bis (3-aminopropyl) Cadverine in barley as well as in powdery mildew (Erysiphae graminis sp Hordei) mycelium, which were reduced in barley leaf on infection with powdery mildew. They opined that the homologous synthesis in barley use S-adenosyl metheonine decarboxylase and spermidine synthase, while in fungus it is different route i.e., Schiff base pathway, could be the reason for variation.

The regulation of DAO in different plants could be different. The diamines oxidase (E.C. 1.4.3.22, 128 KD) has a low rate of catabolising Cad, unlike Put in groundnut (Arachis hypogea).¹⁰¹ It is observed that Put analog 1 aminoxy-β aminopropane (APA) at conc 0.1 mM enhanced the Cad titer in wheat (Triticum monoccum) tissue culture, but beyond that proved to be toxic, which is considered to be unlike to other PA.¹⁰² Whereas, culture trailed for long time, shows elevated activity of ADC as well ODC leading to increase in Put, Cad and spermidine.¹⁰³ This is a serious question that how Put regulating factors affect Cad titer. It is interesting to note that factors affecting Put synthesis induce Cad synthesis remarkably. It could be suggested that probably APA can induce decarboxylases.

Transport of Cadaverine

The basic understanding of diamine transport and transporter are not well elucidated in eukaryotes in general.^104,105 The polyamine transporters have only been characterized in Saccharomyces cerevisiae, Trypanosoma cruzii (TcPAT12).¹⁰⁶ and Leishmania major (LmPOT1)¹⁰⁷ at the cellular level. However, Le Quesne and Fairlamb¹⁰⁸ suggested that transport activity is saturable and requires membrane potential. The Bagni and Pistocchi¹⁰⁹ suggested the possibility of nonpolar transport of Cad through the xylem cells in higher plants. Some transporters are reported in protoplast, vacuole and mitochondria.^110-112 The APC (aminoacid/polyamine/organocation) superfamily transporters are the unique transporter.¹¹³ Generally, it is considered that polyamine transporters are permeases, which are being governed by its concentration. Hasne and Ullman¹⁰⁷ first reported cell surface transporter (LmPOT1) for putrescine, spermidine from Leishmania major. Later the same group reported 2 permeases (Tc POT1.1 and Tc POT1.2). In fact, still the detail of transport of polyamines including Cad either extra/intra cellularaly need to be elaborated¹⁵ and specificaly with the crop plants. In fact this understanding may throw light on the quality of edible parts and the pathogencity, as proper level is essential for bacterial growth.¹¹⁴ The tissue specific transport mechanism i.e., either acropetal in cotyledons to stem could be prominent compared with that of basipetal transport in root tissue.¹³

Under hyperthermic conditions, cadaverine rapidly transported in the basipetal direction, and accumulated in nonheated roots of Mesembryanthemum crystallinum.¹¹⁵ The sensitivity of translocation with temperature was also recorded in vegetable products.¹¹⁶

Despite the de novo synthesis which is considered as the major source of polyamines, transport in and out of the cell, also contributes to polyamine homeostasis. Like all other metabolites/growth regulators homeostasis, a prerequisite for cell growth and development, Cad homeostasis could be possibly by its transport regulation in cells of plants which may be similar to animals.¹¹⁷ The polyamines transport in general plants system is yet to be clearly demonstrated.

Although system of polyamine transporter in lower organism is proposed this has to be tested in higher plants. The six ways of polyamines transport in E.Coli is proposed, viz., uniporter like PotABCD for spermidine, PotFGHI for putrescine, MdtJI for spermidine, Puup for putrescine while some antiporter PotE for Put/ornithine exchanger, CadB for Cad/lysine.¹¹⁸ These all could be operating as per physiological condition of bacteria. However, the putative Cad transporter CadB of E.Coli has been demonstrated.¹¹⁹ To explore the physiochemical responses in yeast, the 9 transporter proteins has been demonstrated playing role.¹²⁰ However, these studies may be extrapolated for understanding of Cad transport in higher plants.

These studies may pave new directions for higher plants transporters functional genomics after having genome maps.

The Cad putative transporter gene structure (Cad operon) and their function/regulation is described.¹²¹ The whole Cad operon consists of CadC (transcription activator of Cad operon), CadB (lysine/Cad antiporter) in CadA (inducible ldc) gene (Fig. 4A). This is found to be inducible with Cad substrate lysine at low pH.¹²² Thus, it is likely that lysine could enhance the expression of CadB and CadA operon involve in LDC coding under acidic condition (Fig. 4B). Soksawatmaekhin and coworkers¹²³ proposed that formation of Cad possibly neutralized the medium extracellularly by the virtue of production of CO₂ and aminopropyle cadaverine and subsequently proton motive force production. It is otherwise when CadBA could not be induced leads to expression of speF-potE operon responsible for ODC and ornithine-putrescine antiporter coding.¹²⁴ These studies also indicates that CadBA operon is responsible for cellular pH control. Because CadB and PotE operon involved in cadaverine and putrescine uptake respectively under neutral pH in polyamine mutants required for growth.¹¹⁹ Soksawatmaekhin et al.¹²³ have identified cadaverine recognition site on CadB based on the information concerning putrescine recognition by PotE. CadB has 6 transmembrane helices with functional amino acid residues at helices II, III, VI, VII, X, and XII. The residues which are involved in either Cad uptake under neutral pH or generating acidic pH leading to synthesis of Cad were mainly localized in the 4 cytoplasmic loops (C2, C4, C5, and C6) (Fig. 4B). In fact, Arg299 involved in the excretion activity of cadaverine localized in loop C5. The amino acid residues involved only in uptake are located in the periplasmic loops (P3 and P6) of the transmembrane segments. This is in contrast to PotE where residues involved only in uptake are generally localized in the cytoplasmic loops of transmembrane segments.¹²⁵ It is also proposed that the SH-group of Cys370 may be also important for both uptake and excretion by CadB.

Figure 4. (A) Pictorial presentation of cad operon having cadC (positive regulator of cad operon) and cadBA genes (cadB = Cad-Lys antiporter; cadA = Lys decarboxylase) [121]. (B) Physiological function of CadB in E.coli, in acidic condition CadB protein functions as electrogenic diamine-amino acid antiporter. In neutral condition, CadB functions as a cadaverine and proton symporter (adopted from 123).

The N-terminal amino acid similarty studies of CadB and PotE protein shows structural and functional homology in both employed in Cad uptake and excretion. Kashiwagi and coworkers¹²⁵ studies in E. coli further demonstrated the Cad pH dependent transport/uptake by CadB transporter which was chopped by a proton uncoupler like carbonyl cyanide m-chlorophenylhydrazone. However, otherwise the Cad uptake or transport under neutral pH condition of a cell remained unaltered inspite of lower level of CadB transcript in polyamine mutant E.coli . The bacterial polyamine mutant studies further substantiates the Cad essentialilty in cell growth.

CadB protein from bacteria shows sequence homology with a amino acid permease (LOC_Os03 g45170.1) in rice identified using TIGR blast and have conserved domain of amino acid impermease2 (Fig. 5). This suggests evolutionary link as well as basic mechanism of uptake and transport similarity in prokaryotic and eukaryotic organisms. The role of this transporter is further established. Amino acid permease gene expression in rice with different developmental stages (using gene investigator software) propound of presence and role of transporter in wide variety of plants, might have dual function, depending on physiological conditions or requirement of plant sustainability (Fig. 6).

Figure 5. CadB protein from bacteria sequence homology with an amino acid permease (LOC_Os03 g45170.1) in rice (using TIGR blast), color shows the bacterial and rice system conserved domain of amino acid impermease2.

Figure 6. Pictorial presentation of Amino acid permease gene (LOC_Os03 g45170) expression in rice at different developmental stages using gene investigator software (www.geneinvestigator.com).

Cad role in growth and development

Cad and Put having two different chemical structures expected to elicit responses differently, but it is found that both elicit similar positive responses on seed germination and in seedling development in chick pea.⁷⁵ Contrary, Grzesik,¹²⁶ observed marked increase in Cad in germinating seed of Helichrysum bracteatum resulted in electrolyte leakage affecting germination. The inhibitory role of Cad is mentioned in opening and closing of stomata in Vicia faba.¹²⁷ The Cad involvement is proposed in rooting in case of soyabean.⁸⁷ Certain negative responses of Cad are also noticed in photosynthetic enzyme like pyruvate orthophosphate dikinase, NADP-malate dehydrogenase in Rumex dentatu¹²⁸ and lipid metabolizing enzyme lipo oxygenase 1.¹²⁹ Interestingly, the Cad did not inhibit lipo oxygenase 2 isozyme in soybean.¹²⁹ These observations suggests Cad inflicting role in energy and C-balance in higher plants. Vassileva and Ignatov¹³⁰ observed alteration in C and N metabolism in root nodules of Galega orientalis at Cad concentration ranging from 0.01–2.0mM, where they reported lower Cad concentration induction of N₂-ase activity.

No effect of Cad on chloroplast outer envelop porins (OEP) involved in transport of triosephosphate, dicarboxylic acids, charged amino acid, sugars, ATP and Pi further indicates specific role in metabolic processes during plant growth. Whereas, Cad effect on bacterial porins are mentioned.¹³¹ In fact, no effect on uricase enzyme in Cicer arientinum, V.faba and T.aestivum leaves¹³² further demonstrates Cad specific role in metabolism of plants. Like Cad checks protein degradation either in dark or in light in Chinese cabbage leaf, while as spermidine and spermine increased protein degradation.¹³³ It is shown that dark induced senescence in rice, the Cad or 1,3diaminopropane was not detected even with HPLC, where as other PA’s were noticed.¹³⁴ The experiments performed in intact leaf of rice during senescence also failed to demonstrate Cad presence.¹³⁵ This is tempting to conclude that Cad endogenous level did not play significant role in controlling dark induced senescence as in case of rice.¹³⁴ However, in some of studies endogenous Cad has been correlated with exo and endo peptidase activity during re-growth of root and stolons of defoliated Trifolium repens.¹³⁶ Desjouis et al.¹³⁶ recorded the inhibition of endopeptidase activity in stolons but not in root except during early periods of growth. The wound injury in chicken pea internode increased Cad level remarkably without affecting spermine and spermidine and demonstrated a close relationship between lignosuberization catalyzed by peroxidase (POD), probably produced under cadaverine oxidation catalyzed by DAO.¹³⁷

Cad titer correlated with development

Infact, the concentration of PAs and inter ratio considered to play important role in plant development.¹³⁸ The Cad titer has been different in different condition depending on plant species suggesting developmental regulation. Cad level declined progressively with seedlings height in Malus hupenhensis.¹³⁹ The downregulation of LDC is also known.⁸² The Cad differential distribution in specific tissue during developmental stages of plants also indicates titer regulation under ontogenic development. Gamarnik and Frydman,⁸⁷ found considerably high Cad in embryonic axes than cotyledons of G.max. The same was high during first 24 h of germination in soyabean, confirmed by application of difluoromethylornithine (DFMO). It is recorded that DFMO (1mM) did not affect other PAs but resulted into abnormal root growth, which was reversed by 1mM Cad suggesting further the Cad dependent root growth⁸⁷ may be plant specific. Cad increases during germination of pea seeds but declined in shoot on prolong period of growth (6–18 d) in light condition. There are some reports that the root may have 70 fold more Cad than leaves that could be due to higher metabolic rate and LDC localization in chloroplast.⁸⁰ On the other hand, the dark grown axes showed higher Cad (2 µmol/g Fr. Wt.).¹⁷ Apart from varied response with physical factors, CaCO₃ or ammonium treatment or K⁺ deficiency in Phaseolus aureus, P. vulgaris or Vicia faba did not change the titer.¹⁷ The tissues dependent Cad titer is also mentioned. Cad was higher in male or female flowers than that of vegetative tissue of Mimordica charantia, suggesting role in sex differentiation.¹⁴⁰ Although, Chen et al.¹⁴¹ have found no correlation between sex reversion and Cad content in cucumber flower, the cultured cucumber cotyledons cells did not show increase in Cad during growth.⁶⁵ Whereas, the tissue specific Cad content is noticed in some cases of cultured cells like Cad increases more during bud differentiation of callus than the root differentiation of callus, but differentiated cell had remarkable higher level than that of non-differentiated callus.⁶⁷ The non-morphogenic de-differentiated cell lines of wheat exhibited low level of Cad than Put.¹⁴²

The ratio of Cad / total PA is negatively correlated with height in crab apple (Malus hupehensis), where as Spd/Cad has been positively correlated.¹⁴³ Cad (0.5, 1.0, 2.0 and 5.0 mMol/L) inhibited rice seed germination and seedling growth, predominately root.¹⁴⁴

The embryonic axes growth in Cicer arientinum is related with increased Cad level, where decrease in Spd and Spm is recoreded.¹⁴² Cad 0.5mM has a full potential to induce flowering in Pharbitis nil cv Kidachi compared with that of Put flowering up to 98% only.¹⁴⁵ The little amount of Cad detected in embryos, but high level in hypocotyl and roots of soyabean, unlike to P.vulgare where gradients of Put, spermidine and spermine increasing from base to shoot apex.¹⁴⁵

Caffaro and Vicente,¹⁴⁶ have demonstrated that Cad along with other PA’s are involved in the flowering processes. These observations suggest that Cad alone may not be affecting physio-chemical process, rather inclusive with other members of polyamines family. The exogenous Cad application induces rooting in the presence of ectomycorrhizal fungi in scot pine¹⁴⁷ and nodule metabolism by changing bacteriods.¹³⁰

Cadaverine in stress modulation and senescence

The few studies are devoted to understand the potential of Cad in stress alleviation in plants (Table 2). The diamine (putrescine and cadaverine) and polyamine (spermidine and spermine) are considered as candidate of the signal systems which is employed in defense system against stress.^148,149

Table 2. Cadaverine marked in plants under stress conditions.

Plant species	Stress condition/Responses	Reference
Brassica napus	Osmotic	89
Brassica napus	Drought	152
Glycine max	Salt	196
Lycopersicon esculantum	Salt	197
Mesembryanthemum crystallinum	Heat, Salt	58,115,166
Oryza sp	Osmotic	72
Oryza sp	Water	156
Pisum sativum	Herbicide	163
Triticum aestivum	Low Temperature, Salt	64,198
Zea mays	Salt	199

In general, PAs carrying cationic charges tend to attach with negatively charged proteins or nucleic acids thereby interfering in possible breakdown and provide stability under stress conditions, and simultaneously suppressing the DNases, RNases and proteases enzymes.¹⁵⁰ PAs are capable to bind A- or B-DNA form, the Cad binding to the sugar-phosphate backbone is proposed thus tendering stability to DNA.¹⁵¹ In stress tolerant plant species, the decreased level of Put are compensated by Spm and Cad. Somehow, the role of Cad in plants under stress, in spite of its massive accumulation in few higher plants,¹⁵² is not well elucidated except a plausible proposal that blocked of Put synthesis might divert the lysine to Cad synthesis for compensating PA’s like in microbial system.¹⁵³

The wide accetance of polyamines including Cad as anti senescence activity^{89,127,152,154} in one hand and no effect on senescence in few cases¹⁵⁵ on the other warrant extensive experimentation to elucidate its role in plant growth in general and under stress in specific. Liu et al.¹²⁷ are also of opinion that the physiological relevance of polyamines increment under stress is not fully explained except that sensitive species deficient in PA as compared with some of the tolerant plants. The same kind of reports in case of Cad is also noted. The Cad also tends to accumulate under osmotic/salt stress like other polyamines.¹⁵² For instance, accumulation of Cad in rice under water stress,¹⁵⁶ and in maize under salt stress, a little increased compared with other PA.¹⁵⁷ Bouchereau et al.¹⁵² have suggested osmotic and salt stress induced production of Cad in higher plants. The rap and tomato leaf disc subjected to 100–300 mM NaCl accumulated Cad, which doubled on the increase in concentration of KCl.⁸⁹ Root Cad increased several times with heat shock and diverts the Cad translocation to shoot.¹⁵⁸ This was unlike to Put which is translocated to leaf tissues.^159-161 Cad increased many fold in ice plants under heat shock.⁵⁸ The studies indicate that Cad synthesis and translocation appears to be heat labile.

The pollutants effects on Cad level is also observed in sensitive and tolerant Phaseolus vulgaris cultivars. For instance, P.vulgaris cv slowianka, a tolerant one exhibited Cad level increase in root and leaves at lower concentration of sulfite, but decreased on increase in sulphite concentration.¹⁶² An herbicide napropamide (10–20 µm) prolonged treatment to pea roots caused 2-fold increase in Cad.¹⁶³ However, there are controversies regarding Cad role in inducing salt tolerance and changes induced in plant metabolism in response to salt stress by Cad.¹⁶⁴

However, Cassan et al.⁷² suggest the Cad role in root growth and osmotic stress reversal in rice seedlings rhizosphere with Azospirillum brasilense Az39 strain promoted in rice seedlings. But, there are few more conflicting reports on rice responses. Such as, Lefevre and group¹⁵⁶ suggested that salinity has no relationship with Cad level in Oryza sativa. Further, Cad absence noticed in rice cell suspension culture and also under K⁺ deficiency.¹⁶⁵ A little amount of Cad found in maize in salt stress.¹⁵⁷ It seems that Azospirillum sp might be contributing to Cad production in rice led to stress mitigation needed further experiments to look for the possibility which might be species and physical condition dependent.

Kuznetsov et al.¹⁶⁶ reported that the addition of cadaverine to the stressed tissues of ice plant suppressed DNA damage and increase the antioxidant enzymes. They further suggested that PAs antioxidant effects could be inducing the SOD expression, possibly by elevating its mRNA transcript. The exogenous Cad differential response on metabolites and enzymes in Indian mustard stressed with salinity is also recorded.¹⁶⁷

The role of other polyamine in anti senescence and anti-stress are quoted widely.^89,127,152 However, the 3 different concentrations (0.2mM, 1mM and 10mM) of Cad tested found ineffective in dark induced senescence in barley.¹⁵⁵ There had been differential responses noticed in number of other cases as well. The lipoxygenase-1 was inhibited by Cad but not lipoxygenase-2.¹²⁹ The Cad had either greater inhibitory response in some cases or almost similar compared with that of Put.¹²⁹

Vassileva and Ignatov¹³⁰ suggested that Put, precursor for Spm and Spd, the inter/intra inhibitory response and interaction could be reverted by Cad. The detail of Cad role on an anti senescence and anti-stress is need to be elucidated vis-à-vis compared with other amines.

Cad Role in Plant Pathogenecity

Polyamines have been implicatd in pathogenecity also depending upon their titer and plant tissues. Legaz et al.¹⁶⁸ have recorded higher SH-Cad in leaf, root and stem of infected plants than non-infected and suggested to be indicator of Ustilago scitaminea causing smut of sugarcane. Cad level did not exhibit any correlation with smut (Ustilago scitaminea) disease development in sugarcane.¹⁶⁹ El-Ghachtouli et al.¹⁷⁰ have demonstrated positive correlation between PA chain length and fungal development. Foster and Walters¹⁷¹ reported that Cad production optimised in in vitro culturing of economically important plant pathogens such as Erysiphe graminis sp hordei and Puccinia hordei on barley, Uromyces on Viciae-fabae and Botrytis fabae on broad fava beans, Podosphaera leucotricha on apples and Phytophthora infestans on potatoes. However, Cad alone showed no effect on powdery mildew and rust infection in barley,¹⁷² where as substitute of Cad does.

The Cad is implicated in growth and development of microbes. The use of DFML causing LDC inhibition leads to inhibition of bacterial growth due to check on peptidoglycon synthesis.¹⁷³ It is observed that both diamines, Cad and Put are essentially required in few pathogenic microbes for their peptidoglycon synthesis. This Cad role might be of clinical significance. Cad blocks the enterotoxins produced by Shigella sp¹⁷⁴ The absence of LDC in Shigella stains noted uniformly.¹⁷⁵ Thus the loss of LDC in Shigella and commercial E.Coli inhibited the enhanced virulence.¹⁷⁴ The inactivation of Cad locus gene (responsible for LDC) in Shigella and enterovasive E.Coli (EIEC) acquires virulence and suggested to be pathoadoptive mutation.¹⁷⁶ Because the restoration of LDC activity after its complementation in negative strain of Cad operon was observed in tissue culture with reduced adherence.¹⁷⁷ Keeping economical significance for number of pathogens pathogenecity in plants are yet to be elucidated in detail with respect to Cad. The differential susceptibility of plant species with various pathogens needs to address with a view to draw a model for possible loss due to disease management.

Cad interaction with other Growth Regulators

The physical property of molecules apart from its titer may also be responsible for variations in physiochemical processes. Chemically, Cad nearer to Put in charge distribution, hence likely to compete for the same site for many metabolic actions like other plant hormones. For instance, Put and Cad inhibit paraquat influx in root of maize intact seedlings. It is shown that Cad inhibits the influx of Put competitively.⁶³ Cross talk between lysine and Cad in the plant is taking place at the uptake and transport system. Lysine enhances the mRNA of CadB¹⁷⁸ but moderately by ornithine where as putrescine transporter Pot E mRNA enhanced moderately by lysine. It is also noticed that Pot E catalyzes exchange of putrescine-lysine in E.coli¹⁷⁹ can be assumed that plants devoid of Cad or having trace amount may be because of less acidic condition or expression/induction of LDC gene/ enzyme protein. The Cad application to cotyledons of Ricinus communis showed decline in the putrescine in sieve tube¹⁸⁰ and a similar response was noticed with spermidine treatment suggests some molecular interaction.

The Cad production in plants varies with ethylene application and implicated in inhibition of ethylene in tomato ripening.¹⁸¹ The possible involvement of ethylene in regulation of Cad level in plants under stress is being already suggested.¹⁸² The ethylene exposure increased Cad production in marigold¹⁵⁸ and in detached leaves of ice plants,⁵⁸ whereas ethylene reduces Put and spermidine (Fig. 2B). It implies that Cad synthesis is also ethylene regulated.^58,158 In Arabidopsis thaliana, the mutations cause dramatic increase in Cad in leaves but not in root.⁵⁸ Kuznetsov and coworkers⁵⁸ have recorded ethylene dependent Cad formation. Further Cad supplemented to cotyledons of Ricinus communis induce decline in putrescine,¹⁸⁰ shows some competition at either synthesis or degradation.

Few chemicals are also known as modulator of Cad synthesis. In Triticum monoccum L. culture, amino-oxy-3aminopropane (0.1 mM) increased Cad compared with that of untreated or treated with 1.0mM.¹⁸³ ABA long exposure of the tissues did not increase Cad in chickpea embryonic axes.¹⁸⁴ Infact, Cad has been considered as signal transducer as well.⁵⁸ Further studies may throw more light on the endogenous interactional behavior of Cad in plant that may be economically significant.

Industrial Application of Cadaverine

In view of emphasis on application of knowledge (s), attempts have been made to use Cad in various industrial purposes. Cad is being used in detection of reactions catalyzed in presence of Ca²⁺ like biotin labeled Cadaverine incorporation in N′-N′-dimethyl casein in Ca²⁺ dependent manner, indicator of the trans glutaminase (Protein-glutamyl transferase) activity.¹⁸⁵ This could be employed to test the enzyme activity during growth period of root and leaves of pea. This has been shown to be potent inhibitor of porins in bacteria, but not chloroplast outer envelope protein-24 (OEP 24), irrespective of homologies among porins of bacteria and mitochondria. This shows precise differentially regulated functionality of the diamines.¹³¹

The Cad and other PA’s present in sugarcane juices involved in binding the phenols, having lower water solubility, thus implicated in development of the color of sugary products, tend to decide the quality.¹⁶⁸ It is worth to explore application of the Cad for removing the phenols and/or any such substance from food products for improving food quality. Further, it also indicates that the genetic manipulation of LDC gene, could be used in developing transgenics and molecular farming of alkaloids and/or Cad. Anabasin production in tobacco hairy root culture has been tried.³⁷ To have commercial production of Cad or related alkaloids in specific plant or species in specific physiological condition could be achieved. The tobacco transgenic with ldc gene elevated expression observed with bidirection to promoters ligated with rbcs promoter of potato.¹⁸⁶ A significant elevation (0.3–1% of dry mass) found in Cad. Pincus et al.¹⁸⁷ opined that Cad might be employed in developing Anti HIV drugs, which needs urgent focus of the scientist for the well being of larger society at present. Even the cancerous cell management could be planned either by regulating Cad titer and/or alkaloid level directly or indirectly by food quality.

Conclusion and Hypothesis

Like other polyamines, Cad influences growth in few cases. The few differential responses could be utilized for plants economical manipulations. Cad presence in plant is scarce in general and under stresses specifically. The pathway of biosynthesis of Cad established seems to be similar both in microbes and animals.⁴⁴

Kamio¹⁸⁸ demonstrated anaerobic bacteria contained with Cad linked with peptidoglycan otherwise gene lacking for the rest of polyamines, this provided uniqueness to microbes. Cad is having some unrelated influences on plant processes for instance, translocation of paraquat in roots of intact maize seedlings,⁶³ making a reason for screening of large plant species for their Cad presence and its relevance.

It is suggested that PA’s profile could be considered in chemotaxonomy for genus Xanthomonas.¹⁸⁹ The disproportional Cad presence/absence, suggest that the Cad can be exclusively used as a chemotaxonomy marker for higher plant species. This requires a great deal of screening of plant family for Cad occurrence.

However, it is also considered that the differential response mediation in different plant system could be due to varied potential of PA induction of phosphorylation of protein,¹⁹⁰ but the same time, it is found that Cad has either little or no effect on phosphorylation of soluble protein in relation to other PA. This indicates Cad potential of regulating the metabolism or related gene expression in specific manner where ever, and could be a process and/or plant dependant. There are cross talk among Cad and other di-tri and tetramine influencing/inducing each other as linked pathway of metabolism. It was opined that synthesis of Cad from lysine, an important and essential amino acid, is channelized in a lesser amount¹⁵² asserts its classification/grouping in growth regulator family. The evolutionary significance for plant development and other functions are yet to be established. However, it is established that Cad is a catabolic product of lysine^3,191 except few cases where ornithine can be used. Based on reports on Cad, having potential of plant growth regulation, many other very important roles in pathogenicity and industrial application (like food industry) need to be elaborated. Recently, the involvement of LDC/Cad in siderophore in microbes are demonstrated.¹⁹² It is likely that the isoform of LDC and Cad may be potentially modulating in plants by alarmone (ppGpp) like nucleotide as proposed in case of microbes.¹⁹³

The PA’s including Cad wide presence is noticed from bacteria to plants, animals and in human. Their various metabolic roles are assigned in regulation of metabolic processes during growth and development of the plants. Further more the PA’s roles are confirmed at the molecular level also. Moreover in recent past PA presence in brain is suggested to be involved in damage repair and cancer prevention.¹⁵ This medical pharmacological aspect needs detail investigation for developing precise, specific aid to ailing people. However, Cad presence in flesh still warrants research to link it not only in terms of pharmacology but also with food quality, shelf life etc.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.