Introduction: Polyamines, Nitrogenous Molecules Related to Several Processes

One of the biggest challenges of modern science is to apply the fundamental discoveries from basic research in providing solutions aiming at increasing food crop yield. Moreover, the increment of the world population has added greater pressure on the demand [1]. Global climate change is expected to intensify the severity and frequency of water stress events and soil salinization worldwide, causing decrease on growth rate and pollen viability, which severely affect crop production [2, 3]. As a result, plant stress physiology research has been widely directed towards the investigation of genetic determinisms underlying stress tolerance of higher plants as well as macroalgae, greatly due to their importance to cover global nutritional needs. In regard with this research area, polyamines (PAs) are relevant molecules [4, 5].

PAs are aliphatic organic compounds with variable hydrocarbon chains and two or more amino groups [6]. PAs have been detected in all cells across all kingdoms [7]. PA biological roles were initially thought to be structural. Their polycationic nature enables the binding and stabilization of anionic macromolecules in the cell. However, later studies revealed that PAs are also involved in fundamental cellular processes such as cell division, differentiation, transcriptional regulation, and translation [8].

Genetic analyses indicate that the most abundant PAs in plants (putrescine (Put), spermidine (Spd), and spermine (Spm)) are required for plant stress tolerance [7, 9, 10]. In addition, these PAs are important sinks of assimilated nitrogen [11]. However, other quantitatively less abundant PAs have shown unique roles in PA functions and stress signaling. Recently, it has been shown that an isomer of Spm (thermospermine (T-Spm)) found in higher plants [7] is not a minor polyamine [12] and it is involved in tolerance responses [13]. In addition, cadaverine (Cad), a formally recognized minor PA [14], is involved in different PA-related signaling processes, modulating plant development and response to external stimulus [15].

Recent and extensive reviews present compelling evidence regarding the participation of PAs in abiotic and biotic stress responses in plants [5, 8]. In reference to salt and drought stress tolerance responses, there is no doubt about the implication of PAs in the physiological processes that leads to plant resilience. However, the nature of the signaling mechanisms and the number of interactions between PAs and other molecules (such as sugars, nitric oxide (NO), ABA, and H2O2) are not only far from being fully elucidated but are also increasing in complexity. Currently, plant tolerance to salt and drought stresses can be achieved by either transgenic approaches regulating PA homeostasis or exogenous addition of individual PAs through the activation of priming phenomena [16]. This is due to the implication of PAs in important pathways of central metabolism and/or modulation of several functions in plant organs such as roots and leaves. Thus, it is important to summarize recent findings regarding the involvement of each PA in drought and salt stress responses.

Brief and Essential Insights in PA Metabolism: Homeostasis Mechanisms

Metabolic studies indicate that the intracellular levels of PAs in higher plants are mostly regulated by anabolic and catabolic processes, as well as by their conjugation to hydroxycinnamic acids (HCAA) [9] and transport by specific membrane transporters [17].

PA biosynthesis is initiated by the synthesis of the diamine Put. In plants, Put is derived from arginine (Arg) through multiple sequential steps catalyzed by the enzymes arginine decarboxylase (ADC), agmatine iminohydrolase (AIH), and N-carbamoylPut amidohydrolase (CPA). Higher PAs Spd, Spm, and its isomer T-Spm are synthesized by the action of aminopropyltransferases (APT) Spd synthase (SPDS), Spm synthase (SPMS), and tSpm synthase (tSPMS, ACL5) which catalyze the addition of aminopropyl groups to Put or Spd, producing Spd, Spm, or T-Spm, respectively. Decarboxylated S-adenosylmethionine (dSAM), a product of S-adenosylmethionine (SAM) decarboxylation by SAM decarboxylase (SAMDC), is the donor of the aminopropyl groups required for PA biosynthesis [6, 18, 19]. On the other hand, there is less information about Cad biosynthesis in plants. It is known that it is mainly synthesized from lysine by lysine decarboxylase (LDC) and homoagmatine or homoarginine to a lesser extent [14, 20].

Amine oxidases (AO) involved in oxidation of PAs are classified in copper-containing amine oxidases (CuAO) and FAD-dependent polyamine oxidases (PAO) [8, 10] releasing in all cases hydrogen peroxide (H2O2). CuAO enzymes preferentially oxidize the primary amino groups of Put and Cad and show less affinity for Spd and Spm [11] and participate in PA final catabolism in peroxisomes and the apoplast [21, 22]. PAOs catalyze the oxidation of higher PAs Spd, Spm, and T-Spm at their secondary amino groups [23]. Classification of PAOs is dependent on whether they catalyze the terminal oxidation of PAs or mediate the PA back-conversion, which is related to the position of the carbon in which PAO is able to oxidize the PA molecule. First group of PAO oxidize Spd and Spm, producing hydrogen peroxide, diaminopropane, 4-aminobutanal (from Spd), and 3-(aminopropyl)-4-aminobutanal (from Spm) [11]. The second group oxidize Spd and Spm (and/or their acetylated derivatives) producing Put and Spd, respectively [24, 25].

In plants, PAs occur in free amine forms but also conjugate to hydroxycinnamic acids to form hydroxycinnamic acid amides (HCAA), in a process catalyzed by N-acyltransferases. Recently, several proteins with Spd N-acyltransferase activity have been reported in Arabidopsis supporting the role of HCAA in pollen tube development [26]. Several years ago, PA conjugates were considered to be inactive forms. However, evidence in the last decade indicates that they are essential for development and biotic interactions [8]. Indeed, an emerging area of research is the characterization of PA conjugates and their pathways involved [8].

PA transport also plays a pivotal role in the regulation of PA cell homeostasis [17]. To date, few PA transporters have been identified in plants, with special attention to rice [27] and Arabidopsis [17]. Recent discovery of PA transporters by genetic analyses has involved the analysis of oxidative tolerance or PA uptake capacity [28]. This investigation opened a new branch of PA research. Interestingly, over the last few years, it was found that transporter AtOCT1 is implied in Cad efflux process [29] and also that Cad flux can be regulated by overexpression of genes encoding transporters (OCT1 included) due to Spm accumulation, proposing a new role for Spm in the regulation of intracellular Cad concentration, which is also redox-dependent [30]. Research on PA transporters will provide deeper insights into the molecular function of PAs and its homeostasis and better understanding into the PA antioxidative properties [17].

Essentially, the PA pool is dynamic and fluctuates over time. PAs undergo rapid exchange through what has been referred to as the “PA cycle,” in which different cycles of biosynthesis and back-conversion lead to the release of H2O2 [9]. This mechanism has been proposed to induce plant stress tolerance [23] and stress signaling affecting multiple cellular compartments [15, 3134]. In agreement with this, recent findings in plants with impaired production of Spd and Spm by SAMDC depletion suggested that SAMDC-mediated higher PA biosynthesis is a putative modulator of the trade-off between stress tolerance and plant growth and developmental traits [34]. Furthermore, Cad has been demonstrated to affect the accumulation of higher PAs in plant tissues [15]; therefore, its contribution to plant stress response cannot be assessed on its own. In any event, it seems reasonable to argue that PA homeostasis modulated by SAMDC, APTs, and AOs is not a simple PA biosynthesis/degradation process.

Interactions of PAs with ROS, NO, ABA, and Other Metabolites

A common acceptance in current research regarding plant stress physiology relates with a number of essential interactions between PAs and signaling molecules such as H2O2, NO, and ABA [3538]. PAs have been related to ROS through H2O2 production via their catabolism pathway [19, 22, 39]. Nonetheless, their cross-talk appears to be far more complex. ROS derived from oxidation of PAs are vital to trigger stress response signaling. However, the size and rate of its accumulation determine cell fate, which means that ROS should not exceed specific thresholds; if so, it shifts the balance between gene expression modification to programmed cell death [40]. Recent findings established that Spd homeostasis by AtPAO3 is involved in ROS production other than H2O2. The ratio of O2 / H2O2 showed to be an important signal in transcription [31] and might be a mediator of PA contribution to plant adaptation under suboptimal conditions [41].

On the other hand, it is widely accepted by PA researchers that PA metabolism and NO biosynthesis are tightly interconnected. NO biosynthesis is stimulated by Spd and Spm in the root tip and primary leaves of Arabidopsis seedlings [42]. A recent study in citrus demonstrated the tissue-specific modulation of PAO expression upon nitro-oxidative stress, after which pretreated plants show increased tolerance to salinity, suggesting that PAs may mediate the link between oxidative and nitrosative signaling [43] and are able to reprogram the nitro-oxidative status as well as the proteome of salinity-stressed plants [44]. It is also known that Spm and Spd modulate the arginine-linked NO synthase and nitrate reductase pathways [45]. NO is filling gaps between several physiological effects of PAs and the amelioration of stress [31, 46, 47], which is essential for further understanding of stress physiology events.

With regard to tolerance mechanisms and stomatal closure, the role of ABA is undeniable, especially during stress response [48, 49]. Interestingly, SPMS is an ABA-inducible gene [50], which is associated with Spm protective role found in Arabidopsis during water stress [51]. However, Put accumulation is also related with increased drought tolerance [52]. Transcriptional upregulation by drought of PA biosynthetic enzymes AtADC, AtSPDS, and AtSPMS and resulting increases in PA content are ABA-dependent [53]. Similar trends were recorded in ADC expression patterns in response to salinity [54] and cold stresses [55]. Conversely, Put was found to promote ABA biosynthesis in response to drought in Lotus tenuis [56]; therefore, a cross-regulation between ABA and Put is not excluded. In support to this, transcriptomic analyses of transgenic SAMDC1 or SPMS overexpressing Arabidopsis plants exhibiting higher Spm levels revealed the transcriptional upregulation of the ABA biosynthesis gene NCED, ABA accumulation, and induction of genes involved in water deprivation and defense responses, including several transcription factors [57]. The relationship between PAs and ABA is significant, and overlapping functions between Put and Spm in regard with ABA have been documented [58].

Another important component under consideration is the role of PAs in central metabolic networks. Recent reports suggest the interplay between PAs and primary metabolism during exposure to stress and/or plant development [5964]. Put, proline (Pro), and GABA are derived from glutamate (Glu), a hub molecule of nitrogen metabolism [59]. Glu signaling impacts amino acid metabolism, which is directly linked to carbon mobilization pathways. In this regard, GABA has been suggested to mediate C/N balance [65, 66] via Glu receptors [67]. PA and GABA accumulation has been reported under both optimum and stressful conditions [68]. Similarly, a metabolic interaction has been detected between PA and sugar metabolism [69]. This is intriguing since sugar signaling is known to be important in the stress response [62, 70, 71]. Apart from known metabolic connections previously reported, PAs have also been implicated in the modulation of metabolite levels for which no metabolic connections are known. Put over-producing Arabidopsis plants revealed the differential regulation of stress-responsive, hormone, and signaling-related genes, involved in the biosynthesis of hormones such as auxin, ethylene, and ABA among others [57]. Overexpression of SPDS induced the transcription of various putative stress-related genes in chilling-stressed transgenic Arabidopsis plants compared with wild-type ones [72]. In addition, transgenic Arabidopsis plants accumulating Spm exhibited modified transcript levels of genes involved in the biosynthesis of jasmonates, salicylic acid and ABA, mitogen-activated protein kinases, receptor-like kinases, and genes linked with calcium regulation [57]. In that sense, recent findings from our group revealed that lack of Spm degradation mechanisms by knocking out PAO4 expression in Arabidopsis leads to an increase in metabolites of central sugar and lipid metabolism such as pyruvate or myo-inositol as well as an enhancement of the antioxidant capacity [61]. In support of this, it was also shown that T-Spm accumulation triggers metabolic and transcriptional reprogramming, enhancing among others, osmoprotectants such as proline, sugars, polyols, and TCA cycle intermediates [64]. In addition, it has been demonstrated that T-Spm modifies the expression of auxin-related genes [33]. Regarding Cad, this PA serves as a critical precursor for the synthesis of secondary metabolites and specific alkaloids involved in defense in Leguminosae as well as several other plants [20]. The identification of PA-regulated downstream targets and the discovery of the interplay between PAs and other stress-responsive molecules have opened new possibilities to investigate the function of individual PAs at molecular level [73]. Therefore, the progressive investigation about the involvement of PAs in several pathways of central metabolism is essential to further understand the role of these molecules under optimum and stress conditions.

Current PA-Related Research Towards Drought and Salt Stress Protection

PAs provide tolerance against several stresses including water and salt stress, through different mechanisms and in relevant species of agronomic use such as maize, wheat, rice, tomato, potato, grape, cacao, or lettuce among others [7483]. This might be the result of their accumulation in the cell providing direct protection against stress or because of PA degradation products which act as signal molecules triggering stress protection. The following sections summarize recent findings regarding PAs and their protective roles against drought and salinity (see Table 1).

Table 1 Selected studies in PA-related research towards drought and salt stress protection employing genetic modification of PA pathway components or exogenous application of PAs

PA Implications in Drought Tolerance

The adaptation of plants to drought involves a series of complex physiological processes and alterations that can take place over the short or long-term (e.g., stomatal closure limiting carbon assimilation) [105]. After drought imposition, plants must coordinate processes to alleviate both cell hyperosmolarity and oxidative stress as well as osmotic imbalance [106]; therefore, physiological alterations at several levels of metabolism must be expected. PA application increases the levels of osmoprotectant molecules and alleviates oxidative damage, which is caused by the stressor and it is additionally involved in other important plant tolerance metabolic processes. Furthermore, a link between PAs and well-known stress-core molecules such as reactive nitrogen and sulfur species has been described.

Exogenous Spm treatment in mung bean conferred tolerance to separate or combined drought/heat stress [84] and salt stress [94] by upregulating the gene expression of important essential antioxidant enzymes (e.g., SOD, CAT) and enhancing the glyoxalase system, which reduced methyglyoxal toxicity, a stress-induced compound [84]. A similar trend was also reported in tomato [85]. Furthermore, recent reports on white clover showed that exogenous application of Spd improves drought tolerance by increasing the level of soluble carbohydrates (WSC) such as sucrose, fructose, and sorbitol as well as dehydrin synthesis [86]. Inhibition of PA biosynthesis reduced cytosolic Ca2+ and antioxidant enzyme activities, while these effects were mitigated by the external application of Spd. Besides this, Spd enhanced the activation of NR and NOS which are the two most known enzymatic sources of NO in the cell. Both NO and Spd enhanced drought stress tolerance by triggering the signaling cascade to enhance the levels of enzymatic antioxidative machinery in a NO-mediated process [87]. Interestingly, it has been recently reported that NO also modulates ABA signaling in Arabidopsis during drought imposition, demonstrating that stomatal closure is not only ABA-dependent but also NO-dependent [88]. Remarkably, PA content increased after H2S treatment thus providing important evidence that another reactive species interacts with PAs, in this case by controlling PA levels under drought conditions. More specifically, it was demonstrated that H2S significantly enhances the tolerance of Spinacia oleracea to drought stress, by changing the transcript levels of genes associated with sugar and PA biosynthesis, which in turn promoted higher sugar and PA accumulation [90]. Recently, it was also shown that drought alleviation by H2S in wheat involves the ABA signaling pathway [91], thereby adding further support to the considerable evidence about the critical role of H2S in physiological and metabolic processes in plants [89, 107].

In addition, some reports have attempted to unravel the involvement of PAs in species- or tissue-specific processes during drought stress condition. A pioneering study on Triticale, a synthetic hybrid of wheat and rye, demonstrated that drought stress tolerance on this species at late developmental stages was linked with a gradual increase in cell wall-bound PAs and a decline in free PAs, thus suggesting a possible role of cell wall strengthening by PAs under abiotic stress conditions [93]. In regard with wheat, it has been found that higher PAs under drought conditions are not only involved in tolerance to stress but also in grain filling [92], which is severely inhibited during water deprivation. More specifically, it was demonstrated that particularly in grains, Spd and Spm significantly increased ABA and zeatin concentrations and decreased the ethylene evolution rate, which promoted wheat grain filling under water deficit conditions, suggesting that this process mediated by hormone interactions is PA-related [92].

PAs and Salt Stress Tolerance

Among the various abiotic stress factors, soil salinization is one of the main harmful factors limiting crop productivity, especially on species highly sensitive to salinity such as cereals [3]. It has been reported that more than 6 % of global land area and 20 % of irrigated lands are affected by this kind of stress, often simultaneously with soil alkalinization, severely reducing plant growth due to concomitant nutrient limitation, ion toxicity, high-osmotic, and oxidative stress [95, 108].

PAs are commonly applied exogenously at a range of concentrations on many plant species, in order to protect plants upon salt stress imposition. Recent findings in cucumber seedlings under salt stress suggested that exogenous Put addition alleviates stress by regulating protein production at transcriptional and translational levels. Interestingly, Put application caused an increase in the levels of endogenous PAs, mainly Spd and Spm, which in turn enhanced fatty acid mobilization that leads to a stabilized photosynthetic apparatus [97]. In addition, omics analyses revealed that Put was able to restore root growth in cucumber seedlings grown under salt stress by enhancing the expression of genes encoding proteins that are involved in defense responses and carbohydrate and amino acid metabolism [98]. Similar findings were also observed after exogenous Spd treatment [99]. Interestingly, another important class of protein which was enhanced by Spd application was proteins related to maintenance of C/N balance [99].

Counteracting positive results on salt stress alleviation obtained by Put application, it has been found that GABA derived from Put degradation ameliorates the deleterious effect of salt stress [100, 101]. However, positive regulation of PA biosynthetic pathway has been reported after GABA exogenous addition to muskmelon seedlings under ionic/osmotic stress [102]. Overall, the implication of Put on salt stress responses is clear, with GABA potentially playing a key role promoting synergistic dynamics with PA levels.

Regarding higher PAs, exogenous application of Spd in zoysiagrass exposed to salt stress caused enhancement of PA biosynthetic pathway and antioxidative enzymatic machinery [103]. Proteomic analysis of tomato seedlings exposed to Spd exogenous pretreatment followed by salt/alkaline stress imposition demonstrated that the protective effect of Spd was mediated by the enhancement of five major categories of proteins involved in energy, sugar, and amino acid metabolism [95]. Later on, a more specific protective effect of Spd was demonstrated on chlorophyll and D1 protein content, which collaborates with the maintenance of photosynthetic apparatus and continuation of photosynthesis rate, thus facilitating the growth of tomato seedlings under salt/alkaline stress [96].

A recent study on Medicago plants under salt stress reported an Spm protective effect due to interactions with epibrassinolide, a plant hormone highly involved in salt stress response. Exogenous addition of epibrassinolide induced PA biosynthetic pathway and concomitant Spm increase caused amelioration of oxidative damage and modulation of Pro levels [104]. In agreement with this, recent findings reported the accumulation of Pro, sugars, and other osmoprotectants, in atpao5 mutants impaired in T-Spm oxidation [64].

The involvement of sugars, amino acid, and oxidative metabolism in the PA signaling pathway after stress imposition has been established. However, further research is imperative in order to fully elucidate the complex physiological responses observed.

Conclusions and Future Perspectives

Drought and salinity lead to yield penalties in modern agriculture. A lot of studies have been conducted to understand and solve the complexity of the processes that occur in plants during stress imposition. In this context, several molecules have been described to play a pivotal role in plant tolerance against stresses, with PAs shaping up as important players. PAs are molecules involved in several fundamental cellular processes, and their production is enhanced under drought and salinity. Remarkably, not only the most abundant PAs (Put, Spd, Spm) are playing a role in stress tolerance but also the quantitatively less abundant ones such as T-Spm and Cad. Different approaches, such as transgenic technologies in the PA pathway and exogenous application of PAs, have been employed to unravel the effects of PAs on drought and salinity. Clearly, there is involvement of PAs in central metabolism and in triggering several mechanisms related to plant stress tolerance (such as through osmoprotection and by inducing the antioxidant machinery). However, a more detailed description on the mechanistic involvement of PAs and the interaction with other molecules to accelerate plants’ tolerance is far from being elucidated because of the complexity of their roles. The dynamic nature of these metabolites, which is related to rapid degradation/interconversion from one to another in the PA cycle, and the interactions between PAs and signaling molecules, such as hormones and reactive species, provide solid evidence on their role in plant stress tolerance. For these reasons, research in that area is ongoing and must to be continued with state-of-the-art approaches such as systems biology technologies, in order to get a better understanding of the contribution of PAs on plant stress tolerance.