Polyamines in response to abiotic stress tolerance through transgenic approaches

Abstract

The distribution, growth, development and productivity of crop plants are greatly affected by various abiotic stresses. Worldwide, sustainable crop productivity is facing major challenges caused by abiotic stresses by reducing the potential yield in crop plants by as much as 70%. Plants can generally adapt to one or more environmental stresses to some extent. Physiological and molecular studies at transcriptional, translational, and transgenic plant levels have shown the pronounced involvement of naturally occurring plant polyamines (PAs), in controlling, conferring, and modulating abiotic stress tolerance in plants. PAs are small, low molecular weight, non-protein polycations at physiological pH, that are present in all living organisms, and that have strong binding capacity to negatively charged DNA, RNA, and different protein molecules. They play an important role in plant growth and development by controlling the cell cycle, acting as cell signaling molecules in modulating plant tolerance to a variety of abiotic stresses. The commonly known PAs, putrescine, spermidine, and spermine tend to accumulate together accompanied by an increase in the activities of their biosynthetic enzymes under a range of environmental stresses. PAs help plants to combat stresses either directly or by mediating a signal transduction pathway, as shown by molecular cloning and expression studies of PA biosynthesis-related genes, knowledge of the functions of PAs, as demonstrated by developmental studies, and through the analysis of transgenic plants carrying PA genes. This review highlights how PAs in higher plants act during environmental stress and how transgenic strategies have improved our understanding of the molecular mechanisms at play.

Introduction

Plant development and productivity are negatively affected by environmental stresses. The major abiotic stress factors limiting crop productivity and plant growth are soil salinity, exposure to high and low temperatures, and drought. In the past decade, the average yields of major crops have been reduced up to 70% due to abiotic stresses.¹ On the other hand, the increasing world population has added more pressure on the demand for increased crop yields, creating an urgent need to cultivate stress-tolerant crops with increased productivity.² Agriculture around the globe is facing great challenges due to climate change with more erratic climate and with it, an increasing effect of abiotic stress factors. Plants are able to sense environmental changes, to respond to them, adapt and survive. Such changes under stress occur at cellular, physiological, biochemical, and molecular levels. The expression of a variety of genes is induced by different stresses in a wide diversity of plants.³ However, the stress-tolerant plants can alter their cellular mechanism and metabolic pathways to counteract the created imbalance under environmental stresses.⁴ The ability to survive of stress-tolerant plants depends on the proper maintenance of the structural and functional integrity of cells.⁵ Due to the complex nature of stresses, multiple sensors, rather than a single sensor, are responsible for a plant’s response to stress. In general, various nitrogen-containing compounds accumulate in plants in response to environmental stress such as amino acids (arginine, proline), amino acid-derived compounds, amides (glutamine, asparagine), ammonium, quaternary ammonium (glycinebetain), and polyamines (PAs).^6-9 In addition to the maintenance of cellular functions and osmotic balance, these amino acids and their derivatives, including PAs, protecting cellular components, such as membrane protein complexes, by acting as molecular chaperons or regulators.^10-12 Diverse stress conditions activate both acclimatization and adaptation techniques of plants to deal with abiotic stresses by using general and conserved stress response mechanisms. This is exemplified by Arabidopsis thaliana, which uses a common signal transduction pathway that involves the use of PAs as secondary messengers.¹³

PAs are low molecular weight, non-protein polycations at physiological pH with a strong binding capacity to negatively charged DNA, RNA, and different protein molecules, and the ability to stabilize membrane structures.^10,14 They regulate many physiological, growth, and developmental processes in organogenesis, embryogenesis, flower initiation and development, leaf senescence, fruit development and ripening, and abiotic and biotic plant stress responses.^15-18 The induced accumulation of putrescine (Put) in response to potassium deficiency was among the first reports to demonstrate a link between PAs and abiotic stress.¹⁹ Since then, a large number of reports have shown the relationship between PA accumulation and the activities of their biosynthetic enzymes during and after stresses.^11,13,18,20 In addition to common PAs, several uncommon PAs also accumulate under different stresses.^21-23

The availability of cloned PA biosynthetic genes from various sources and subsequent transfer of arginine decarboxylase (ADC), S-adenosylmethionine decarboxylase (SAMDC), and spermidine (Spd) synthase (SPDS) showed improved environmental stress tolerance in various transgenic plants. Transgenic plants overexpressing different PA biosynthetic genes showed that the accumulation of different PAs allowed these plants to develop multiple stress tolerance.^12,24-27 This review summarizes the relation of PA biosynthesis in abiotic stress responses, their pathways during stress, modulation of PA metabolic pathways by transgenic technology and finally transcriptomic and metabolomic approaches to unravel the key functions of different PAs in the regulation of abiotic stress tolerance. Nevertheless, the precise molecular mechanism(s) by which PAs control plant responses to stress stimuli remain largely unknown. Recent studies indicate that PA signaling is involved in direct interactions with different metabolic routes and intricate hormonal cross-talks to elucidate the proposed model of environmental stress tolerance capacity of PAs.

Polyamine Biosynthetic Pathway under Abiotic Stresses

The biosynthesis and degradation pathway of PAs in plants is well documented in Figure 1. Put is synthesized either directly from decarboxylation of the amino acid L-ornithine by ornithine decarboxylase (ODC; EC 4.1.1.17) or indirectly, by the decarboxylation of l-arginine by ADC (EC 4.1.1.19) via agmatine.²⁸ The product of ADC is agmatine, which is converted into Put through an intermediate N-carbamoylputrescine. Conversion of agmatin into Put requires two distinct enzymes: agmatin iminohydrolase (EC 3.5.3.12) and N-carbamoylputrescine aminohydrolase (EC 3.5.1.53). The increased synthesis of PAs under different stresses is activated by the ADC pathway.^18,29 The ODC pathway is more active in the early stages of plant growth, development, organ differentiation and reproductive stage. A few plant species, including Arabidopsis thaliana, lack the ODC pathway.^14,30

Figure 1. Polyamine biosynthetic and main catabolic pathways in Plants. ACC, 1-aminocyclopropane-1-carboxylic acid; ADC, Arginine decarboxylase; DAO, Diaminoxidase; ODC, Ornithine decarboxylic acid; PAO, polyamineoxidase; SAM, S-adenosylmethionine; SAMDC, S-adenosylmethionine decarboxylase; SPDS, Spermidine synthase; SPMS, Spermine synthase (ref. 18, after modification).

Spd and Spm are synthesized by the successive attachment of aminopropyl with Put first to synthesize Spd and then Spd to synthesize spermine (Spm).^28,31 These reactions are catalyzed by aminopropyltransferases such as Spd synthase (SPDS, EC 2.5.1.16) and Spm synthase (SPMS, EC 2.5.1.22). Aminopropyl is formed due to the decarboxylation of S-adenosylmethionine (SAM) by S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50). SAM is produced from the amino acid l-methionine and ATP by S-adenosylmethionine synthetase (SAMS, EC 2.5.1.6). Aminopropyl transferases belong to a group of widely distributed enzymes and their genomic organization has been studied in maize (Zea mays).^31,32 Activation of the ADC pathway and accumulation of higher PAs, Spd and Spm, was hypothesized under different environmental stresses.^23,33-36 A halophytic plant, Mesembryanthemum crystalinum, and A. thaliana showed a similar response under high salinity stress.^36,37 Recent reviews concluded the positive role of PAs in abiotic stress tolerance.^2,9,38,39 PA catabolism efficiently regulates the level of free PA in the cell which can perform an important physiological role under normal and stress situations.^40,41 Diamine oxidase (DAO; EC 1.4.3.6) and PA oxidase (PAO; EC 1.5.3.3) are responsible for oxidizing the diamine Put and PA Spd and Spm, respectively.^22,42

Polyamines in Response to Different Abiotic Stresses

Metal stress

Potassium (K) is an important macronutrient and common stress-related factor carrying vital functions in plant metabolism, growth and stress adaptation. However, studies related to this type of stress are often performed on leaves and/or seedlings, as the external symptoms of deficiency become acute. The accumulation of Put in the leaves of K-deficient barley (Hordeum vulgaris) plants,¹⁹ and the subsequent studies, established the specific role of Put in maintaining a cation-anion balance in plant tissues.¹⁸ The accumulation of different PAs and activated enzymes under different stress conditions are listed in Table 1. Watson and Malmberg⁴³ investigated the regulation of ADC activity in relation to Put content during K⁺ deficiency stress in A. thaliana. The effects of cadmium (Cd) on Put accumulation by the activation of ADC was confirmed in bean (Phaseolus vulgaris) seedlings by using ADC enzyme inhibitor (L-α-difluromethylarginine, i.e., DFMA) and ODC enzyme inhibitor (L-α-difluromethylornithine, i.e., DFMO).⁴⁴ Later on, different studies on the exposure of heavy metals such as Cd, copper (Cu), nickel (Ni), and zinc (Zn) also demonstrated the accumulation of different PAs and increased ADC activity.^7,45 PA metabolism and the antioxidant property of Spm was proposed as a heavy metal stress-protecting mechanism. Different studies suggested the function of Spm as an antioxidant in protecting tissues from metal-induced oxidative damage to a certain extent.⁴⁵ Heavy metal stress, Cu- and chromium (Cr)-induced Put and Spd accumulation in plants afforded them protection against oxidative damage.⁴⁶

Table 1. Abiotic stress-induced polyamine (PA) accumulation and biosynthetic enzyme activation in plants.

Abiotic stress	Plant species	Accumulated PA(s)	Enzyme activated	Ref. #
Aluminum	Catharanthus roseus	Put	-	65
Cadmium	Avena sativa Helianthus annuus Phaseolus vulgaris	Put Put, Spd, Spm Put	ADC ADC, ODC ADC	44 45 44
Chromium	Raphanus sativus	Put	-	76
Cold	Cicer arietinum	Put, Spd	-	82
Copper	Helianthus annuus Raphanus sativus	Put, Spd, Spm Put, Spd	ADC, ODC -	45 46
Heat	Arabidopsis thaliana Gossypium hirsutum	Put, Spd NorSpd, NorSpm	ADC, SAMDC -	71 68
Lead	Potamogeton cripus	Put	ADC, PAO	75
Mercury	Chlorogonium elongatum	Put	-	74
Osmotic	Cicer arietinum Lupinus luteus Sorghum bicolor Zea mays	Put, Spd Put, Spd Spd, Spm Spd, Spm	- ADC - -	79 58 78 78
Potassium	Arabidopsis thaliana Hordeum vulgaris	Put Put	ADC -	43 19
Salt	Aeluropus littoralis Arabidopsis thaliana Brassica campestris Helianthus annuus Lupinus luteus Malus domestica Oryza sativa Oryza sativa Oryza sativa Zea mays	Spd Spd, Spm Put, Spd Put, Spd Put, Spd Put Put Put Spd, Spm Spd, Spm	- ADC ADC, ODC - ADC ADC ADC - - -	61 37 62 80 58 59 50 81 51 60

ADC, Arginine decarboxylase; NorSpd, Norspermidine; NorSpm, Norspermine; ODC, Ornithine decarboxylase; PAO, Polyamine oxidase; Put, Putrescine; SAMDC, S-adenosylmethionine decarboxylase; Spd, Spermidine; Spm, Spermine.

Salinity, osmotic, heat, and/or cold stress

The first report of the accumulation of Put and Spd in oats (Avena sativa) cells and protoplasts by exposure to various osmotica (sorbitol, mannitol, proline, betaine, sucrose, etc.), initiated PA research in response to osmotic stress.⁴⁷ Osmotic stress induced an increase in ADC transcript level, supporting the ADC activation pathway during stress.⁴⁸ Osmotic stress induced an increase in the level of Put and diaminopropane (Dap) with a decrease in the level of Spm in rape (Brassica napus) plants, explained by the role of Spm in post-translational regulation of ADC.⁴⁹ The accumulation of PAs in response to saline stress^49-51 and their exogenous application help to overcome the harmful effects of NaCl stress was reported in rice (Oryza sativa) seedlings.^52-54 Several other studies showed that exogenous application of different PAs helped to overcome the stress effect to some extent.^55,56 The effect of salinity stress on the activation of ADC, as well as its increased transcript level was observed in NaCl-tolerant rice cultivars exposed to 150 mM NaCl for 12 h.⁵⁷ Salt and osmotic stress induced the accumulation of Put and Spd and increased ADC activity in the roots and leaves of lupine (Lupinus luteus) seedlings and in apple (Malus domestica).^58,59 Increased accumulation of Spd and Spm with increase tolerance to salt stress was observed in maize seedlings and in flowers and flower stocks of A. thaliana while growing constantly in 200 mM NaCl.^37,60 Increased levels of Spd and Spm, as well as increased ADC and SPDS expression levels, were detected in A. thaliana by reverse transcriptase polymerase chain reaction (RT-PCR). In most of the studies concerning salt stress, induced PA accumulation was due to the activation of ADC. NaCl stress induced a reduction in the level of Spm in the shoots of Mediterranean salt grass (Aeluropus littoralis), caused by the degradation of Spm to higher PAs.⁶¹ A study of ADC and ODC inhibitors shed light on the induction of the ADC pathway and on the accumulation of Put and Spd under stress: although the activation of both ADC and ODC in dicotyledonous plant, turnip (Brassica campestris) was reported, but there was no inhibitor study or mutant analysis to confirm the observations.⁶² A study of the PA level and PA enzyme activity during salt stress in PA-deficient mutants of A. thaliana demonstrated the decreased formation of Put due to lower ADC activity, which ultimately led to reduced salt tolerance.⁶³ Drought stress in rice also increased bound and free Put with increased resistance.⁶⁴ PAs improve K/sodium (Na) homeostasis in barley seedlings by regulating ion channel activities and thus improving stress tolerance.⁶⁵ The translocation and accumulation of Put and other PAs in an organ-specific manner after external application during salt stress afforded protection to rice.⁶⁶ Put imparted protection to salt stress by improving water relations and nutrient imbalance in cucumber (Cucumis sativus).⁶⁷

Under heat stress, heat-tolerant plants showed increased levels of higher PA (Spd and Spm) pools.^22,23 Moreover, long chain uncommon PAs (norspermidine, norspermine, and caldopentamine) were also noticed during exposure to heat stress, similar to a thermophilic bacterium, Thermus thermophilus.^23,68 Only heat-tolerant rice cv “N22” accumulated norspermidine and norspermine after heat stress. ADC and PAO activities increased to a large extent in heat-tolerant ‘N22’ than in the heat-sensitive cultivar ‘IR8’. Exogenous application of 1 mM Spd and 1 mM Spm helped to recover the heat shock (50 °C for 2 h) in mung bean (Vigna radiata) seedlings.⁶⁹ Spd and Spm content and SAMDC activity increased after rice seedlings were chilled at 5 °C for 3 d.⁷⁰ Put, Spd, and Spm content increased and SAMDC2, SPMS, and ADC2 genes were induced during 35 °C heat shook for 1 h in A. thaliana. It was also reported that the SPMS transgenic plants were protected from heat shock damage similar to exogenously applied Spm effect by the expression of heat shock protein genes (HPS101, HPS90, and HPS70).⁷¹ The effect of drought stress at supraoptimal temperatures on free proline and PA levels were compared in wild and proline-overproducing transgenic soybean (Glycine max).⁷² Similarly, the relationship of PA and proline in drought and heat stress tolerance responses in proline over-producing transgenic tobacco (Nicotiana tabacum) plants was explained: increased Spm level was one factor that imparted stress tolerance.⁷³ These studies indicate that manipulation of proline synthesis also affects the level of PA by developing stress tolerance and adaptation. Stress induced accumulation of proline and different PAs were known to improve osmotic stress tolerance and maintaining ionic balances in cellular environment.⁷⁷

Molecular Cloning and Expression of Polyamine Biosynthetic Genes Induced under Abiotic Stress

A large number of genes encoding key enzymes for the PA biosynthesis pathway have been cloned and characterized from various plant species following the cloning of oats (Avena sativa) ADC.⁸³ The cDNA of ADC, ODC, SAMDC, SPDS, and SPMS cloned from different plants are listed in Table 2. Differential expression of two genes encoding ADC (ADC1 and ADC2) was observed in A. thaliana in response to environmental stresses. The expression of ADC2, SPDS1, and SPMS was strongly induced by several abiotic stresses (dehydration, high salinity, K⁺ deficiency) while ADC1 was mainly induced by cold.^84-87 However, no changes in SPDS2 expression were detected in response to any stress. SAMDC2 expression was induced mainly by cold and salt stress whereas SAMDC1 was induced by cold stress in A. thaliana cell suspensions.⁸⁸ Three cDNAs (MdSPMS-1, MdSPMS-2, and MdSPMS) with high homology to SPMS of A. thaliana were isolated and characterized from apple.⁸⁹DAO cDNA has been isolated, sequenced and compared from pea (Pisum sativum)⁹⁰ whereas PAO cDNA was isolated and compared from maize.⁹¹ Database searches within the A. thaliana genome sequences showed the presence of a gene (AtPAO1) encoding a putative PAO with 45% amino acid sequence identity with maize PAO. The correlation of physiological and biochemical responses of abiotic stress tolerance with increased PA biosynthesis at its transcriptional level is well documented.^{13,32,57,92,93} The expression of these genes under different abiotic stresses was induced and showed a differential response in terms of mRNA induction and accumulation depending on the type, duration and intensity of the stress, the plant species, and other factors. Moreover, transcriptomic, metabolomics, and genetic approaches have implicated the key functions of different PAs in the regulation of abiotic stress tolerance at the global level while cross-talking between different stress hormones and metabolic pathways.^10,13,94 Previously, it was suggested that PAs can act by stabilizing membranes, scavenging free radicals, affecting nucleic acids and protein synthesis, RNase, protease and other enzyme activities, and interacting with hormones, phytochromes, and ethylene biosynthesis.^95,96 However, recent studies focused on the molecular mechanism of PA signaling and their integration with other metabolic pathways during abiotic stress tolerance. The expression of several genes involved in PA biosynthesis was strongly induced by one or more abiotic stresses as well as by abscisic acid (ABA)-induced stress in A. thaliana.⁸⁵ The dehydration stress induced expression of ADC2, SPDS1 and SPMS as ABA-dependent response was observed in a mutant study of A. thaliana.⁸⁶ However, the accumulation of Put and the induction of genes involved in Spd and Spm biosynthesis did not affect the content of both PAs. This could be due to the translational and post translational regulation of SAMDC, a key enzyme in PA biosynthesis.⁸⁶ PA degradation by PAOs and generation of hydrogen peroxide (H₂O₂) under stress is important for protecting stressed cells by lignification and cross-linking extensions in response to stress and wounding.^97,98

Table 2. List of plant-cloned PA biosynthetic genes.

PA biosynthetic genes	Source plants	Accession number	PubMed No. or Ref. No.
Arginine decarboxylase (ADC)	Arabidopsis thaliana	U52851	8756495
	Avena sativa	X56802	2266946
	Brassica juncea	AF22009 / AF220098	12060267
	Citrus trifoliata	HQ008237	21282323
	Dianthus caryophyllus	U63832	99*
	Glycine max	U35367	100*
	Malus domestica	AB181854	15723827
	Nicotiana tabacum	AF321137	15032880
	Oryza sativa	NM001063230 / NM001058553 / AY604047	18089549 18089549 16769152
	Pisum sativum	Z37540	7548836
	Pringleaantis corbutica	AY337606 / AY337607	15527979
	Prunus persica	AB379849	18996450
	Solanum lycopersicon	L16582	8022938
	Vitis vinifera	X96791	1042065
Ornithine decarboxylase (ODC)	Capsicum annuum	AF480882	14972797
	Datura stramonium	X 87847	8660289
	Glycine max	AJ563382	15763662
	Malus domestica	AB181855	15723827
	Nicotiana tabacum	D89984	9869416
	Nicotiana glutinosa	AF323910	11736657
	Oryza sativa	NM001070362 NM001053389	18089549 17210932
	Prunus persica	AB194103	16510395
	Solanum lycopersicon	AF030292	9733552
	Triticum aestivum	HM770451	21192794
	Zea mays	NM001148682	19936069
S-Adenosylmethionine decarboxylase (SAMDC)	Arabidopsis thaliana	Y07765	11139406
	Brassica juncea	U80916 / X95729	9390449
	Catharanthus roseus	U12573	101*
	Dianthus caryophyllus	U94786	9289746
	Hordeum chilense x Triticum turgidum	X83881	8639739
	Ipomoea batatas	AF188998	12060284
	Ipomoea nil	U64927	102*
	Malus domestica	AB077441/AB077442	15781000
	Oryza sativa	Y07766 AB122089	11139406 15215597
	Phaseolus lunatus	AB062360	12060229
	Pisum sativum	U60592	11925048
	Solanum tuberosum	Z11680 S74514	1450379 7948879
	Triticum aestivum	AF117660	92*
	Vicia faba	AJ250026	103*
	Vitis vinifera	AJ567368	16982115
Spermidine synthase (SPDS)	Arabidopsis thaliana	AB062360	9517003
	Coffea arabica	AB015599	104*
	Cucumis sativus	AY646352	106*
	Hyoscyamus nigra	AB006691	9517003
	Malus domestica	AB072915/AB072915	12655406
	Nicotiana sylvestris	AB006692	95170
	Oryza sativa	AB098063	15310079
	Pisum sativum	AF043108/AF043109	10344199
	Solanum lycopersicon	AJ006414	105*
Spermine synthase (SPMS)	Arabis gemmifera	AB076744	12655134
	Malus sylvestis	AB204521	16182474

^*PubMed number of the published article is not available in NCBI site, but papers are available as in text reference.

Polyamine Accumulating Transgenic Plants with Improved Abiotic Stress Tolerance

Modification of the PA level by transgenic approach and study of the role of PA in response to several stresses has been analyzed. Manipulation of the PA level in several plants may lead to improved plant tolerance against multiple environmental stresses are listed in Table 3. Different levels of mRNA accumulation of oat ADC, increased ADC activity and accumulation of PAs at different levels were observed in tobacco, rice, eggplant (Solanum melongena), and wheat (Triticum aestivum).^{23,57,107,109,124} Transgenic plants were generated by expressing the ADC gene of oats and datura (Datura stramoniun) under the control of different constitutive (maize ubiquitin 1) and inducible (tetracycline and ABA) promoters, and transgenic plants showed tolerance to salt and drought with an increased accumulation of Put, Spd, and Spm.^24,26,109 The accumulation of Put as well as enhanced tolerance to dehydration, freezing and salt stress was observed in transgenic A. thaliana.¹¹⁰ Overexpression of ADC of Poncirus trifoliata (PtADC) in A. thaliana showed increased synthesis of Put and enhanced tolerance to high osmotic, drought and cold stress.¹¹¹

Table 3. List of transgenic plants encoding PA biosynthetic genes.

Name of PA genes	Source Organisms	Transgenic plants	Accumulated PA	Tolerance developed	Ref. No
ADC	Avena sativa	Oryza sativa	Put	Salinity	24
ADC	Datura stramonium	Oryza sativa	Put, Spd, Spm	Drought	26
ADC	Avena sativa	Solanum meloangena	Put, Spd, Spm	Salinity, drought, low and high temperature, heavy metal	108
ADC	Avena sativa	Triticum aestivum	Put, Spd, Spm	Drought	109
ADC-2	Arabidopsis thaliana	Arabidopsis thaliana	Put	Drought	10
ADC	Avena sativa	Arabidopsis thaliana	Put	Drought, freezing	110
ADC	Poncirus trifoliata	Arabidopsis thaliana	Put	Osmotic, drought, cold, oxidative	111
Anti ACC	Dianthus caryophyllus	Nicotiana tabacum	Spd, Spm	Oxidative	114
ODC	Mus musculus	Nicotiana tabacum	Put	Salt	121
SAMDC	Hordeum chilense x Triticum turgidum	Oryza sativa	Spd, Spm	Salt	25
SAMDC	Homo sapiens	Nicotiana tabacum	Put, Spd	Salt, drought, fungal wilt	113
SAMDC	Dianthus caryophyllus	Nicotiana tabacum	Spd	Salt, cold, acidic, oxidative	112
SAMDC	Dianthus stramonium	Oryza sativa	Spd, Spm	Drought	122
SAMDC	Saccharomyces cerevisiae	Solanum lycopersicon	Spd,Spm	Heat, oxidative	27
SAMDC	Malus domestica	Nicotiana tabacum	Spd	Chilling, salt, osmotic	123
SPDS-1	Cucurbita ficifolia	A. Arabidopsis B. thaliana	Spd	Chilling, salt, drought	116
SPDS-1	Cucurbita ficifolia	Ipomoea batatas	Spd	Salt, drought	117
SPDS-1	Malus domestica	Pyrus communis	Spd, Spm	Salt, osmotic	118
SPDS-1	Malus domestica	Pyrus communis	Spd	Heavy metals, oxidative	119

ACC, Aminocyclopropane carboxylate; ADC, Arginine decarboxylase; ODC, Ornithine decarboxylase; Put, Putrescine; SAMDC, S-adenosylmethionine decarboxylase; SPDS, Spermidine synthase; Spd, Spermidine; Spm, Spermine.

As SAMDC is one of the key regulatory enzymes in the biosynthesis of PAs, in order to better understand the effect of regulation of PA biosynthesis on the tolerance of different types of stress such as salt, drought, and low and high-temperature stress in rice, tobacco, and tomato (Lycopersicon esculentum), the SAMDC gene of different sources were introduced by Agrobacterium tumefaciens and transgenic plants showed increased environmental stress tolerance and accumulation of higher PAs than non-transgenic plants.^25,27,112 Different SAMDC transgenes of tritordeum (Hordeum chilense × Triticum turgidum), carnation (Dianthus caryophyllus), and yeast (Saccharomyces cerevisiae) showed an inducible and constitutive type of expression under different stresses. Transgenic rice plants containing a tritordeum SAMDC and induced with 50 µM ABA produced a 3- to 4-fold increase of Spd and Spm than unstressed control plants under salt stress and afforded protection to transgenic plants after 150 mM NaCl stress in a re growth recovery study.²⁵ Transgenic tomato plants constitutively expressing the SAMDC gene of yeast accumulated 1.7- to 2.4-fold higher levels of Spd and Spm than wild-type plants under temperature stress (38 °C for 1 h) and showed tolerance to this stress, enhanced antioxidant enzyme activity and low membrane lipid peroxidation.²⁷ Constitutive overexpression of human SAMDC in tobacco gave rise to an increment in Put and Spd levels that led to tolerance to salt and osmotic stress.¹¹³ On the other hand, tobacco plants transformed with antisense ACC synthase or ACC oxidase from carnation showed higher SAMDC activity as well as higher Put and Spd levels, tolerance to oxidative stress, high salinity and low pH.¹¹⁴ In tobacco, constitutive overexpression of carnation SAMDC resulted in the accumulation of total PAs and generated broad spectrum tolerance to abiotic stresses.¹¹²A. thaliana plants constitutively overexpressing a mustard SAMDC exhibited an increased level of Spd and Spm and tolerance to salt, drought, and oxidative stresses.¹¹⁵

Overexpression of the SPDS gene in A. thaliana, sweet potato (Ipomoea batatus), and pear (Pyrus communis) transgenic cell lines showed an increased titer of Spd as well as increased tolerance to different abiotic stresses compared with non-transgenic plants.^116-118A. thaliana plants overexpressing the SPDS cDNA from melon (Cucurbita ficifolia) exhibited a significant increase in SPDS activity as well as Put, Spd, Spm concentration, and tolerance to chilling, freezing, salinity, hyper-osmosis, and drought.¹¹⁶ Moreover, overexpressed SPDS transgenic plants of A. thaliana showed high level expression of various stress-responsive transcription factors such as DREB1A, DREB1B, DREB2B, and stress protective proteins rd29A.¹¹⁶ Overexpression of the apple SPDS gene in pear showed altered PA levels as well as increased tolerance to multiple abiotic stresses.¹¹⁹ Functional analyses of stress tolerance genes identified PAs and proline as molecular chaperones and key protective elements in drought, salinity and temperature stress tolerance.^42,120

Knowledge of the molecular mechanisms governing plant responses to abiotic stresses has increased considerably during the last decade and it is clear that protective mechanisms and metabolic networks connected with several upstream and downstream processes involved in stress tolerance are interconnected. Different abiotic stresses in plant is commonly known to alter osmotic potential and in accumulation of reactive oxygen species (ROS), while PAs may act as osmotic regulators and scavengers of ROS by overexpressing ADC in transgenic A. thaliana.¹²⁵ Exogenous application of PAs (Put, Spd, Spm) in A. thaliana induced nitric oxide (NO) production and transgenic A. thaliana overexpressing rat nitric oxide synthase (NOS) showed enhanced tolerance to dehydration stress (10 d) and 200 mM NaCl stress (2 d) compared with nontransgenic plants.¹²⁶ Genetic engineering of several metabolic pathways of several compatible solutes such as proline or glyceinbetaine for abiotic stress tolerance is also important.^127-130 Abiotic stress tolerance is a complex phenomenon involving the adaptation, perpetuation, and responsive attitude of stress tolerance in a cyclic crosstalk by the control of several metabolic pathways in which PAs mediate signal transduction.^42,73,75,131

All these studies support the view that increasing PA biosynthesis could be a good strategy in transgenic research in response to plant stress and to improve the tolerance of stress-sensitive plants against adverse environmental conditions. It is clear that PAs play a central role in enhanced tolerance to adverse abiotic stresses. However, overexpression of heterologous genes only provides limited information about the importance of endogenous PA synthesis and the function of endogenous enzymes during salt and drought stresses. Studies using endogenous genes and mutants with reduced enzyme activity might help to understand the mechanism of action of PA metabolism in different stress responses.

Conclusions and Future Perspectives

Genetic manipulation leading to increased PA biosynthesis is a potential strategy to study the roles of PAs in a plant’s responses to abiotic stress and to improve their tolerance against adverse environmental conditions. Abiotic stresses are the primary cause of plant losses worldwide, and thus approaches employing genetic modification aimed at overcoming severe environmental stresses need to be quickly implemented in association with molecular-assisted traditional breeding and metabolomics.¹³² A large amount of research data indicates that overexpression of PA biosynthetic genes play an important role in environmental stress tolerance and could be exploited in biotechnological programs to produce stress-tolerant plants. Future strategies should be based on a detailed knowledge-based framework of PA metabolism using PA biosynthetic genes and their regulation by employing different types of stress-inducible promoters and transcription factors. A concrete understanding of the genetic engineering of PA biosynthetic genes, a biotechnological strategy, may be of great importance in solving future problems related to environmental stress-resistant plants using transgenic strategies.¹³³

Glossary

Abbreviations:

ACC

1-aminocyclopropane-1-carboxylic acid

ADC

arginine decarboxylase

Cad

cadaverine

DAO

diaminooxidase

Dap

1,3-diaminopropane

Nor-Spd

norspermidine

Nor-Spm

norspermine

ODC

ornithine decarboxylase

PA

polyamine

PAO

polyamineoxidase

Put

putrecine

SAM

S-adenosylmethionine

SAMDC

S-adenosylmethionine decarboxylase

Spd

spermidine

Spm

spermine

SPDS

spermidine synthase

SPMS

spermine synthase

10.4161/gmcr.28774

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

Acknowledgments

Malabika Roy Pathak wishes to thank Prof Bharati Ghosh for developing research interest on polyamines and the Desert and Arid Zone Sciences Program, Arabian Gulf University, Kingdom of Bahrain.