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. 2011 Feb 1;6(2):243–250. doi: 10.4161/psb.6.2.14317

Polyamine metabolic canalization in response to drought stress in Arabidopsis and the resurrection plant Craterostigma plantagineum

Rubén Alcázar 1,#, Marta Bitrián 1,#, Dorothea Bartels 3, Csaba Koncz 1, Teresa Altabella 2, Antonio F Tiburcio 2,✉
PMCID: PMC3121985  PMID: 21330782

Abstract

In this work, we have studied the transcriptional profiles of polyamine biosynthetic genes and analyzed polyamine metabolic fluxes during a gradual drought acclimation response in Arabidopsis thaliana and the resurrection plant Craterostigma plantagineum. The analysis of free putrescine, spermidine and spermine titers in Arabidopsis arginine decarboxylase (adc1–3, adc2–3), spermidine synthase (spds1–2, spds2–3) and spermine synthase (spms-2) mutants during drought stress, combined with the quantitative expression of the entire polyamine biosynthetic pathway in the wild-type, has revealed a strong metabolic canalization of putrescine to spermine induced by drought. Such canalization requires spermidine synthase 1 (SPDS1) and spermine synthase (SPMS) activities and, intriguingly, does not lead to spermine accumulation but to a progressive reduction in spermidine and spermine pools in the wild-type. Our results suggest the participation of the polyamine back-conversion pathway during the drought stress response rather than the terminal catabolism of spermine. The putrescine to spermine canalization coupled to the spermine to putrescine back-conversion confers an effective polyamine recycling-loop during drought acclimation. Putrescine to spermine canalization has also been revealed in the desiccation tolerant plant C. plantagineum, which conversely to Arabidopsis, accumulates high spermine levels which associate with drought tolerance. Our results provide a new insight to the polyamine homeostasis mechanisms during drought stress acclimation in Arabidopsis and resurrection plants.

Key words: Arabidopsis, Craterostigma plantagineum, drought, polyamines, polyamine oxidase, abiotic stress

Introduction

Drought stress severely affects plant growth and crop productivity.1 Drought stress induces an extensive transcriptional reprogramming in plants, which involves ABA-dependent and ABA-independent pathways.2,3 Eventually, drought induces the accumulation of different osmolytes, like sugars, proline and polyamines (PAs), to cope with dehydration conditions.4–6 Polyamines (PAs) are small aliphatic amines implicated in a wide range of environmental stresses.7–9 Remarkably, drought stress induces changes in PA titers, which broadly correlate with drought resistance traits.8,10,11 Putrescine (Put), spermidine (Spd) and spermine (Spm) are the most abundant PAs and are all derived from arginine or ornithine. The ornithine pathway is constituted by a single gene (ODC; EC 41117), which decarboxylates Orn to produce Put.7 The biosynthesis of Put from Arg requires the activity of three consecutive enzymes: arginine decarboxylase (ADC; EC 41119), agmatine iminohydrolase (AIH; EC 353.12) and N-carbamoylputrescine amidohydrolase (CPA; EC 3.5.1.53). The Arabidopsis genome contains two genes coding for arginine decarboxylase (ADC1 and ADC2), whereas AIH and CPA are found as single copy genes.4,12,13 ODC orthologous genes have not been identified in A. thaliana.14 Hence, PA biosynthesis in this model species relies on the ADC pathway. Conversion of Put to Spd and Spm requires successive addition of aminopropyl moieties by Spd synthase (SPDS; EC 25116) and Spm synthase (SPMS; EC 25.1.16), respectively. The donor of aminopropyl groups is decarboxylated S-adenosylmethionine (dcSAM), which derives from decarboxylation of S-adenosylmethionine (SAM) by SAM decarboxylase (SAMDC; EC 4.1.1.50).15 The Arabidopsis genome carries two genes coding for SPDS (SPDS1, SPDS2), one SPMS (SPMS) and at least four SAMDCs (SAMDC1–4).16,17 The aminopropyl transferases SPDS1, SPDS2 and SPMS are constituted in a macromolecular protein complex, which is predicted to aid an effective substrate channeling preventing the loss of PA intermediates, reducing the transit time between active sites of aminopropyl transferases in consecutive reactions, and avoiding substrate competition with other enzymatic reactions.16 However, so far there is no evidence for an effective Put to Spm canalization during stress responses in plants. The homeostasis of PAs depends not only on their biosynthesis, but also on terminal catabolism and back-conversion of higher molecular weight PAs to their precursors. Put and other diamines are substrates of diamine oxidases (DAO; EC 1436), whereas tri- and tetra-amines, like Spd and Spm, are substrates of polyamine oxidases (PAO; EC 1.5.3.3).7 DAOs catalyze the oxidation of Put and cadaverine to 4-aminobutanal and ammonia, releasing H2O2 in the reaction.18 There are at least 12 DAO-like genes in the Arabidopsis genome, but only ATAO1 has been so far characterized.19 A number of plant PAOs are involved in the terminal catabolism of Spd and Spm, producing 1,5-diazabicyclononane, 1,3-diaminopropane and H2O2. One of the best characterized PAOs from this class is a maize PAO (ZmPAO).18 A second group of PAOs resemble the mammalian Spm oxidases (SMO; EC 1533), which catalyze the back-conversion of Spm to Spd.20,21 The Arabidopsis genome carries at least four genes encoding PAOs (AtPAO1–4). AtPAO1 and AtPAO4 catalyze the back-conversion of Spm to Spd, but conversely to mammalian SMO, they show strong preference for non-acetylated PAs.21 Also, AtPAO1 and AtPAO4 show differential substrate affinities. Hence, AtPAO1 shows strong substrate preference to Spm, whereas AtPAO4 has higher affinity to recognize thermospermine (tSpm) and norspermine.22 AtPAO2 and AtPAO3 are both involved in back-conversion reactions from Spm to Spd, and then to Put.20,22 Coupling PA biosynthesis with PA back-conversion would complete a full PA recycling-loop.

To gain insight to the transcriptional and posttranscriptional regulation of PA biosynthesis under a gradual drought acclimation response, we have studied the levels of free Put, Spd and Spm in adc1–3, adc2–3, spds1–2, spds2–3 and spms-2 mutants and wild-type Arabidopsis plants exposed to a progressive drought stress, and compared to the expression profiles of the entire PA biosynthetic pathway. Accumulation of PAs used as precursors of higher molecular weight PAs and changes in the products of their corresponding enzymatic reactions has allowed the identification of a Put to Spm metabolic canalization in response to drought. A number of drought tolerant species have been used for studying the molecular basis of desiccation tolerance, including the South African resurrection plant Craterostigma plantagineum as the best characterized example.23,24 In this work, we have also analyzed the PA profiles of C. plantagineum in response to drought stress and compared them to those found in the desiccation intolerant species A. thaliana. Overall, our data provide an insight to the PA metabolism fluxes occurring during drought.

Results

Transcriptional profiles of PA biosynthesis genes under drought stress.

In this work, we have exposed A. thaliana plants to progressive drought stress acclimation by withholding water for several days. The expression of ADC1, ADC2, SPDS1, SPDS2, SPMS, ACL5, SAMDC1 and SAMDC2 has been analyzed after 0, 2, 4 and 6 days of water deficit. The expression of drought-inducible genes RD29A and RD22 was used as control to monitor the stress conditions. As shown in Figure 1, the expression of RD29A and RD22 increased after two days of drought treatment, suggesting that increased ABA levels, to which RD29A and RD22 respond, has already occurred at the first time point of analysis.2,25 A second but more moderate increase in RD29A and RD22 expression was observed after 6 days of treatment. As shown in Figure 1, the expression of ADC1, ADC2, SPDS1, SPDS2, SPMS and SAMDC1 increased during the drought exposure and showed similar kinetics to ABA-inducible RD29A and RD22 genes (Fig. 1). Among all genes analyzed, ADC2 exhibited the highest increase in expression (10-fold after 6 days). Only two of the eight PA biosynthetic genes analyzed (ACL5 and SAMDC2) were different from RD29A/RD22 expression kinetics. The expression of ACL5, which synthesizes thermospermine (tSpm) from Spd,26,27 increased 6-fold after 2 and 4 days and dropped to basal levels after 6 days of treatment. A significant repression in SAMDC2 expression was also observed after 2 days of treatment, followed by a continuous increase up to 1.5-fold on day 6. Overall, these results indicate that most of the PA biosynthetic genes follow significant increases in expression and similar expression kinetics under the imposed drought conditions, similarly to the drought-responsive RD29A and RD22 genes. Our results are consistent with an involvement of ABA signaling in the transcriptional regulation of PA biosynthesis under a gradual drought acclimation response (Fig. 1), which was also previously reported for plants exposed to more severe drought stress.28

Figure 1.

Figure 1

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Transcriptional regulation of genes acting in the polyamine (PA) biosynthetic pathway under drought stress in Arabidopsis. Four-week-old plants were exposed to gradual drought stress for several days. Real time RT-PCR measurement of transcript levels of PA biosynthetic genes ADC1, ADC2, SPDS1, SPDS2, SPMS, ACL5, SAMDC1, SAMDC2, Polyamine oxidase 2 AtPAO2, Deoxyhypusine synthase DHS, RD29A and RD22 was performed after 0, 2, 4 and 6 days of drought treatment. ADC, Arginine decarboxylase; ACL5, Acaulis 5; dcSAM, decarboxylated S-adenosylmethionine; SAMDC, S-adenosylmethionine decarboxylase; SPDS, Spermidine synthase; SPMS, Spermine synthase; PAO, Polyamine oxidase.

PA levels in PA-biosynthetic mutants under drought stress.

adc1–3 and adc2–3 mutants were previously characterized in reference 29. In this work, we have characterized new SPDS1, SPDS2 and SPMS mutant alleles in the Arabidopsis accession Col-0 (spds1–2, spds2–3 and spms-2), which were isolated from the Cologne collection of T-DNA insertion lines.30 T-DNA insertions were found in exons 6, 1 and 5 of SPDS1, SPDS2 and SPMS, respectively, which lead to a strongly attenuated expression of their respective transcripts (Sup. Fig. 1). These PA biosynthetic mutants and wild-type plants were used to analyze the free Put, Spd and Spm levels after 0, 2, 4, 6, 10 and 16 days of drought treatment (Fig. 2). The Put levels increased by 1.8-fold after 2 days of drought stress in wild-type plants. This observation is consistent with previous reports showing that Put accumulation occurs under drought stress.7,10 Remarkably, much higher Put levels were observed after 2 and 6 days in the spds1–2 mutant compared to the wild-type. SPDS1 encodes one of the two Arabidopsis SPDS genes (SPDS1 and SPDS2), which utilizes Put as precursor of Spd, in an enzymatic reaction which incorporates an aminopropyl moiety derived from dcSAM to Put. The accumulation of the precursor (Put) in spds1–2 mutant suggests that SPDS1 is involved in the conversion of Put to Spd during the drought stress response. Interestingly, Put accumulation in spds1–2 overlapped with the accumulation of ADC1 and ADC2 transcripts (Fig. 1), thus suggesting a correlation between ADC1 and ADC2 upregulation and Put levels. On the other hand, even though the expression of SPDS2 was increased after 2 days of treatment (Fig. 1), the spds2–3 mutant did not show significant Put accumulation or reduced Spd levels (Fig. 2). This observation suggests that SPDS2 has a very limited contribution to the Put to Spd conversion during drought. Therefore, our data is consistent with a prominent role of SPDS1 during dehydration.28 A significant increase in Put levels was also observed in spms-2 mutant after 4 days of drought treatment. SPMS catalyzes the conversion of Spd to Spm, and is encoded by a unique gene in Arabidopsis.16 Thus, accumulation of Put in spms-2 is likely due to an over-accumulation of Spm precursors (Put and Spd; Fig. 2). Interestingly, although both adc1–3 and adc2–3 mutants showed much lower free Put concentration than wild-type plants in the absence of stress (day 0), Put content in adc1–3 and adc2–3 mutants reached similar levels as in wild-type after 2 days of drought treatment. This observation indicates that ADC1 and ADC2 genes act redundantly to maintain free Put levels similar to the wild-type during drought stress. However, we cannot exclude that the distribution of Put in different tissues may differ between adc1–3, adc2–3 and wild-type plants.

Figure 2.

Figure 2

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Polyamine profiles under drought stress in polyamine biosynthetic mutant and wild-type Arabidopsis plants. Free putrescine, spermidine and spermine levels were analyzed in Arabidopsis wild-type, adc1–3, adc2–3, spds1–2, spds2–3 and spms-2 mutants after 0, 2, 4, 6, 10 and 16 days of drought treatment. Values are the mean from three biological replicates ±standard deviation (SD). DW, dry weight.

The levels of Spd and Spm were also analyzed in all PA biosynthetic mutants. Although SPDS1 and SPDS2 transcription is increased during drought acclimation, we detected a general trend of decreased Spd and Spm titers. Interestingly, we found a significant increase in Spd concentration in the spms-2 mutant after 2 and 6 days of drought treatment compared to wild-type. Remarkably, the two peaks for Spd content overlapped with the higher ADC1, ADC2 and SPDS1 expression levels (Figs. 1 and 2).

The accumulation of Spd in spms-2 mutant is consistent with SPMS mediated conversion of Spd to Spm. Nevertheless, drought stress does not lead to Spm accumulation in the wildtype. In addition, the spms-2 mutant showed a dramatic reduction in the product of its enzymatic reaction (Spm) consistent with the absence of gene redundancy for Spm biosynthesis.16,26,27

Overall, we conclude that a metabolic canalization from Put to Spm occurs under drought stress, and conversely this does not lead to increases in Spd or Spm levels in the wild-type.

ADC and SAMDC activities under drought stress.

As shown in Figures 1 and 2, drought stress induces an increase in the expression of ADC coding genes and accumulation of Put. To test whether ADC expression and Put content correlates with ADC activity, we monitored the decarboxylation of Arg by measuring the release of radiolabeled CO2 from [14C]-Arg during 24 h. As shown in Figure 3, an increase in ADC activity could be detected after 2 h of drought treatment and reached a maximum of 10-fold increase after 24 h of drought exposure. These results indicate that ADC expression correlates with its enzymatic activity and the accumulation of Put under drought stress.

Figure 3.

Figure 3

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ADC, SAMDC, DAO and SMO enzymatic activities under drought. Wild type Arabidopsis plants exposed to drought stress were used for the analysis of arginine decarboxylase (ADC), S-adenosylmethionine decarboxylase (SAMDC), diamine oxidase (DAO) and spermine oxidase (SMO) activities at different time points of 24 h drought treatment. Values are the mean from three biological replicates ±SD.

Transcription of SAMDC1, whose enzyme catalyzes the decarboxylation of SAM to dcSAM required for Spd and Spm biosynthesis, showed more moderate but still significant increase under drought stress (Fig. 1). Hence we tested whether changes in SAMDC activity could also be observed during drought acclimation by measuring the release of radiolabeled CO2 from the decarboxylation of [14C]-SAM by SAMDC. As shown in Figure 3, SAMDC activity did not differ significantly during the dehydration period and showed an average value of 0.07 ± 0.01 pmol CO2·h−1·gprot.−1 Therefore, we concluded that SAMDC activity was not affected by drought treatment.

Polyamine oxidation during drought stress.

PA oxidation by diamine oxidases (DAOs) and polyamine oxidases (PAOs) contributes to PA homeostasis.18 Put is a known substrate for diamine oxidases (DAO), whose enzymatic reactions oxidize Put to 2,4-diaminopropane (Dap). DAO activities were measured by detection of radiolabelled [14C]-Dap derived from the oxidation of [14C]-Put. DAO activity was detectable in Arabidopsis and increased after 30 min of treatment, showing a maximum of three-fold increase after 1 h followed by a progressive reduction to basal levels (Fig. 3). Our results suggest that Put oxidation by DAOs participates in a primary response to drought.

Our data provided evidence for the occurrence of a Put to Spm canalization under drought, which intriguingly does not lead to Spm accumulation but to a progressive reduction in Spm and Spd levels. We tested the hypothesis that an increased Spm catabolism by Spermine oxidase (SMO) would contribute to reduction of Spm and consequently Spd levels during drought stress. Since all PAO genes (AtPAO1–4) characterized so far in Arabidopsis are involved in the back-conversion of Spm to PA precursors,20–22 and none reported in the terminal catabolism, we have measured specific Spm oxidase (SMO) activities in Arabidopsis using radiolabeled [14C]-Spm and detection of [14C]-1,5-diazabicyclononane derived from the SMO enzymatic reaction. Interestingly, SMO activity was detectable in Arabidopsis, although SMO encoding genes have not yet been reported in this species. However, SMO activity did not change significantly during the drought treatment, showing an average value of 83.5 ± 6.8 pmol 1,5-diazabicyclononane gprot 1h−1. Thus, although Arabidopsis has SMO activity, it is unlikely that the terminal catabolism of Spm is responsible for a progressive reduction of Spd and Spm pools, since SMO activity stays constant during drought stress. In Arabidopsis, PAOs AtPAO2 and AtPAO3 are known to be involved in the back-conversion of Spm to Put via Spd.20–22 The expression of AtPAO2 and AtPAO3 is induced by ABA treatment,20 which suggests a potential role in the PA back-conversion pathway during drought. According to Takahashi et al.22 the expression of AtPAO2 is much higher than AtPAO3 in all tissues tested. Therefore, we analyzed transcript levels of AtPAO2 to assess its potential contribution to regulation of PA back-conversion and PA homeostasis during drought stress. As shown in Figure 1, AtPAO2 is upregulated and shows a similar expression kinetics as the ABA-inducible RD29A and RD22 genes during drought stress. Hence, our results support an active role of the back-conversion pathway during dehydration, which could explain the absence of Spm accumulation even in the presence of a strong Put to Spm metabolic canalization.

Deoxyhypusine synthase expression during drought.

Spd is required for the NAD-dependent formation of deoxyhypusine in the eukaryotic translation initiation factor 5A (eIF-5A), which is catalyzed by the deoxyhypusine synthase enzyme DHS.31 eIF-5A is highly conserved and an essential protein in all organisms from archaebacteria to mammals.32,33 To examine whether deoxyhypusination would also contribute to a reduction in Spd levels, we have followed transcriptional response of DHS during drought acclimation in Arabidopsis. Interestingly, transcript levels of DHS followed the same kinetics of upregulation as as those of ABA-inducible RD29A and RD22 genes during drought stress (Fig. 1). These results suggested activation of deoxyhypusination, which might contribute to Spd consumption during drought.

PA levels in C. plantagineum under drought stress.

To compare the regulation of PA homeostasis of Arabidopsis to a desiccation tolerant species, the levels of free Put, Spd and Spm were analyzed in Craterostigma plantagineum plants exposed to drought stress for 4 days. As shown in Figure 4, the levels of free Spd and Spm progressively increased up to 3-fold and 8-fold, respectively, during 96 h of drought treatment. Remarkably, a gradual decrease in Put levels accompanied Spd and Spm increases during the first 24 hours of drought treatment. However, a 2.3-fold increase in Put content could be observed after 96 h of treatment. The early drought adaptation response in C. plantagineum thus implies a stimulation of Spd and Spm biosynthesis with concomitant reduction of Put precursor levels. These observations also suggest the occurrence of a Put to Spm canalization in C. plantagineum that conversely to Arabidopsis, leads to a significant 8-fold accumulation of Spm levels which correlate with drought tolerance traits.

Figure 4.

Figure 4

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Free putrescine (Put), spermidine (Spd) and spermine (Spm) levels in C. plantagineum plants exposed to drought stress conditions for 0, 1, 2, 4, 8, 24, 72 and 96 hours. Values are the mean from three biological replicates ±SD. DW, dried weight.

Discussion

Changes in PA levels occur in response to a variety of abiotic stresses.7–11 Arabidopsis thaliana is the only plant species, in which a full functional compilation of the PA biosynthetic pathway has been achieved.4 Genetic and genomic studies performed in Arabidopsis support a prominent role of PA biosynthetic pathway in stress responses and have provided a major advance in our understanding of PA functions PA homeostasis is a dynamic process, which involves sequential biosynthesis of Put, Spd and Spm, as well as the terminal catabolism of Put and Spd/Spm by DAO and PAO activities respectively, and back-conversion of Spm to Spd and Put.7,9 The complexity in the regulation of PA homeostasis limits the identification of PA fluxes during the stress response by analyzing PA titers, because some of the PA intermediates might not accumulate. In this regard, Panicot et al. reported the occurrence of a PA metabolon constituted by the aminopropyltransferases SPDS1, SPDS2 and SPMS, which would allow an efficient canalization of Put to Spm. In this work, we provide evidence that such canalization indeed occurs during drought stress in Arabidopsis. The PA biosynthetic mutants spds1–2 and spms-2 show higher accumulation of PA precursors (Put and Spd) during drought treatment. By contrast, the spds2–3 mutant does not show accumulation of Put, which is consistent with a prominent role of SPDS1 in the drought response.28 Most of the PA biosynthetic genes follow an expression kinetics similar to the ABA-inducible RD29A and RD22 genes (Fig. 1).25 Thus, our results are in agreement with a prominent role of ABA in controlling transcription of genes implicated in PA biosynthesis during drought stress.28 An exception is the gene ACL5, which does not follow such ABA response kinetics. ACL5 does not interact either with other components (SPDS1, SPDS2 and SPMS) of the PA metabolon. Therefore, it is likely that ACL5 does not participate in a potential Put to tSpm canalization process.16 Although Put to Spm canalization occurs during drought stress, Spm levels do not increase but rather are progressively reduced during the dehydration period (Fig. 2). We hypothesized that an enhanced terminal catabolism by Spm oxidation (SMO) to 1,3-diaminopropane and 1,5-diazabicyclononane could contribute to reduced Spm levels during dehydration. However, we did not detect significant changes in SMO activities under dehydration conditions (Fig. 3). This indicates that higher Spm oxidation is unlikely a reason for reduction of Spm levels during drought in Arabidopsis.

A number of PAOs have been implicated in the PA back-conversion pathway in Arabidopsis and, unlike in mammals, plant PAOs do not use acetylated derivatives.21 AtPAO2 catalyzes the back-conversion of Spm to Put and, interestingly, its expression is upregulated by drought stress in a similar fashion as RD29A and RD22 (Fig. 1). A number of other AtPAO genes were shown to be upregulated by ABA treatment.20 Previous observations and our results support a role for the PA back-conversion in the drought stress response. Coupling Put to Spm canalization with the back-conversion pathway would complete an effective PA recycling-loop. Each step in the PA back-conversion pathway releases one molecule of H2O2, which may contribute to ROS signaling. Remarkably, ROS signaling is tightly linked to the control of PA homeostasis and ABA-mediated stress responses, in which PAs participate.18,34 Stomatal closure is one of such ABA-mediated physiological responses to drought, which is tightly linked to ROS signaling and NO production.35,36 Apoplastic amino oxidases are sources of ROS production18 and ABA activates Put catabolism and H2O2 production during stomatal closure in Vicia faba guard cells.37 PAs are reported to promote the production of NO in this species38 and regulate stomatal responses by reducing their aperture and inducing closure.37,39

Notably, Put oxidation by DAOs is also increased during the early stages of drought treatment in Arabidopsis (Fig. 3). This observation also supports a model which predicts that a PA recycling-loop occurring under drought stress serves as amplification of ROS signaling by recurrent generation of H2O2 that may contribute to ROS-mediated drought stress responses.40 The study of the differences in drought tolerance between species allows the identification of molecular mechanisms underlying such variations. In this regard, the levels of free Put, Spd and particularly Spm at the end of the drought treatment were much higher in the desiccation tolerant C. plantagineum than in Arabidopsis. This is consistent with previous reports showing a correlation between PA levels and stress tolerance traits in different species.7 The high Spm titers observed in C. plantagineum after 4 days of drought treatment are in agreement with the occurrence of a conserved Put to Spm metabolic canalization in this species. Hence, it is likely that Put to Spm metabolic fluxes are evolutionary conserved mechanisms between desiccation tolerant and intolerant species, whereas tolerance traits correlate with the capacity of the plant to adapt to high Spd/Spm titers. Indeed, it has been reported that osmotic stress induces higher Spd and Spm titers in drought tolerant wheat cultivars (Yumai No. 18) than in the drought-sensitive ones (Yangmai No. 9 cv).41 Also, inhibition of SAMDC activity by methylglyoxal-bis-(guanyl-hydrazone) aggravated the osmotic stress induced injury in wheat.41 In vetiver grass exposed to moderate osmotic stress conditions, an increase in free and conjugated Spd and Spm levels in leaves correlated with plant survival.42 In drought tolerant gravepine cultivars, the levels of Put and Spd are significantly increased after drought treatment compared to drought sensitive ones. Drought tolerance also correlated with a differential increase in ABA levels between cultivars.43 In Theobroma cacao, ABA and drought stress induced the expression of TcODC, TcADC and TcSAMDC, and increased Spd and Spm levels which correlated with changes in stomatal conductance.44 In rice, drought stress induced ADC, SAMDC and SPDS activities together with an increase in Put, Spd and Spm contents which correlated with yield ratios.45 Also in rice, the overexpression of Datura stramonium SAMDC lead to an increase in Spm which facilitated the recovery from drought.46 On the other hand, the overexpression of the Datura stramonium ADC in this species lead to higher levels of Put, Spd and Spm, together with an enhanced drought tolerance.47 In pear, overexpression of the apple SPDS lead to increased Spd and Spm titers which correlated with an improved tolerance to osmotic stress.48 In Arabidopsis, very high Put levels induced by genetic engineering were sufficient to promote tolerance to drought stress49 whereas the spms/acl5 double mutant showed enhanced susceptibility to drought, which could be rescued by exogenous application of Spm.50,51 In tomato and other species, high PA levels also correlate with stress tolerance and enhance fruit quality traits.52–54 Overall, evidences point to a requirement of Spd/Spm for a proper dehydration response, consistent with the occurrence of an evolutionary conserved Put to Spm canalization process.

Alternative mechanisms to PA back-conversion, which may contribute to the decrease of free Spd levels in response to drought involve the activation of deoxyhypusination, as suggested in this work (Fig. 1). In addition to their free forms, PAs also occur in plants in conjugates with hydroxycinnamic acids which are referred to as hydroxycinnamic acid amides (HCCAs).7 A gene encoding a Spd hydroxycinnamoyl transferase (SHT) has recently been characterized and suggested to participate in the formation of tricoumaroyl-, tricaffeoyl- and triferuloyl-Spd in the tapetum of Arabidopsis anthers.55 Nonetheless, further studies are necessary to evaluate possible roles of HCCAs in the control of PA homeostasis in response to stress.

Materials and Methods

Plant growth and drought stress treatments.

Seeds were sown in 7 × 7 cm pots containing a mixture of soil and vermiculite [3:1 (v/v)] and stratified in the dark at 4°C for 72–96 h. Arabidopsis plants were grown under standard conditions with 8 h/16 h light/dark cycle, at 100–125 µmol of photons m−2s−1 of light intensity and 21 ± 2°C. Drought treatments were applied to 4 weeks old plants grown in soil by withholding watering up to 16 days. Desiccation of C. plantagineum was performed as described by Bartels et al.56

Measurement of transcript levels.

Total RNA was isolated from leaves of Arabidopsis plants at the indicated time points using the TRIzol reagent (Invitrogen) and further purified with RNeasy Plant Mini Kit columns (Qiagen). One microgram of total RNA was treated with amplification grade DNase I (Invitrogen). cDNA was synthesized using SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative real-time PCR with the SYBR Green I dye method was performed using a Eppendorf Mastercycler ep realplex detector system (Eppendorf). Standard curves for targeted genes were performed from 1:2 serial dilutions of cDNA templates and used for quantification. ADC1, ADC2, SPDS1, SPDS2, SPMS, ACL5, SAMDC1 and SAMDC2 primer sets were described before in reference 28. RD29A, RD22 and DHS primer sequences are (RD29A: 5′-GAA GTT TGC TTC AAT GCT AGG TTA CTC and 5′-TCG TCA CGG CAG ACT CTG TT; RD22: 5′-GAA GTA CCC ATT CGC GGT GT and 5′-TTT AGC TCG CAT CCC GTT CT; DHS: 5′-CGA AGC TGT CCA TGC AAA TCC and 5′-GAC ACG GCT TCA TCA GGG C). PCR conditions were as follows: 95°C 2 min, followed by 40 cycles (95°C, 15 sec; 60°C, 30 sec; 68°C, 20 sec). qRT-PCR measurement of transcript levels were performed in triplicates from two independent experiments.

Polyamine analyses.

PAs were analyzed by high-performance liquid chromatography (HPLC) separation of dansyl chloride-derivatized PAs. The extraction and determination methods have been previously described in reference 57. The analyses were performed in triplicate in three independent experiments.

Identification of spds1–2, spds2–3 and spms-2 mutants.

The spds1–2, spds2–3 and spms-2 Arabidopsis thaliana mutants in Col 0 were identified by PCR-based screen of 90,000 T-DNA-tagged lines using gene and T-DNA-specific primer sets.30 The following primers were used for screen: SPDS1 (5′-TTC TCG GAG ATA TTC ACC AGA GCA ATA ACC and 5′-TCC TCC AGA TTA GTT TTC TTT CCC TTT TCA), SPDS2 (5′-AAA CCC TAA TCT CTT ACT CAC TGT CTC TCT and 5′-GAT TTC TCT TCT CTT TTC TAG TTG GCT TTC), SPMS (5′-CTC TCC GTT TAC GAC CAG TAT ACA G and 5′-AGG GAT ATG GTA GAG CCA AAC AGA AG) in combination with the T-DNA-specific primers FISH1 5′-CTG GGA ATG GCG AAA TCA AGG CAT C and FISH2 5′-CAG TCA TAG CCG AAT AGC CTC TCC A. DNA sequencing of left borders identified the precise positions of the T-DNA insertion. Homozygous plants were identified by PCR and confirmed by segregation analyses in MSAR plates containing hygromicin 15 mg/l to select for the T-DNA encoded antibiotic resistance marker. All heterozygous mutants showed 3:1 segregation on selection media consistent with the occurrence of single T-DNA insertions. RT-PCR expression analyses of SPDS2 and SPMS were performed with primers sets previously described in reference 28. Primers for SPDS1 RT-PCR analysis were: 5′-GTT CCA ACA TAC CCC AG and 5′-TGC TGG AGC TCT CGT CAA TTG GG.

Measurement of ADC, SAMDC, DAO and SMO activities.

Measurement of ADC, SAMDC, DAO and SMO activities were performed in 4-week-old plants exposed to drought conditions for 24 h as described in Alcazar et al.28 ADC, SAMDC and DAO activities were determined as previously described in reference 58.60. SMO enzyme activity measurements performed as follows: 500 mg leaf tissue per sample was frozen in liquid nitrogen and homogenized in 5 ml of extraction buffer containing 100 mM sodium phosphate buffer (pH 70), 5 mM 2-mercaptoethanol and plant protease inhibitor cocktail (Sigma). Extracts were centrifuged at 13,000 rpm at 4°C for 15 min and supernatants used for measurement of SMO activities. Equal loads of total protein extracts quantified by Bradford were mixed in technical triplicates with 0.2 µCi of a 1:5 dilution of [14C] Spermine tetrahydrochloride (Amersham) in 5 mM Spm. SMO reactions were performed for 30 min at 37°C and stopped by addition of 100 µl 20% sodium carbonate. Radiolabelled 1,5-diazabicylononane was extracted by addition of 900 µl of toluene to each sample followed by mixing and centrifugation at 12,000 rpm at room temperature. 400 µl of toluene was recovered and mixed with 3 ml of scintillation cocktail. Radioactivity (dpm) of each replicate was measured for 10 min in a Packard 1,500 scintillation counter. All measurements were performed with three independent biological triplicates per time point of analysis.

Acknowledgements

Our research was supported by grants from the Ministerio de Educacion y Ciencia, Spain (BIO2005-09252-C02-01 and BIO2008-05493-C02-01), CSD2007-00036 and the Comissionat per Universitats i Recerca (Generalitat de Catalunya, SGR2009-1060). R.A., D.B., C.K., T.A. and A.F.T. also acknowledge grants-in-aid from COST-Action FA0605.

Abbreviations

ABA

abscisic acid

ACL5

acaulis5

ADC

arginine decarboxylase

AIH

agmatine iminohydrolase

CPA

N-carbamoyl putrescine amidohydrolase

DAO

diamine oxidase

Dap

1,3-diaminopropane

dcSAM

decarboxylated SAM

ODC

ornithine decarboxylase

PAO

polyamine oxidase

Put

putrescine

SAM

S-adenosyl methionine

SAMDC

S-adenosyl methionine decaboxylase

ROS

reactive oxygen species

SMO

spermine oxidase

Spd

spermidine

SDPS

spermidine synthase

Spm

spermine

SPMS

spermine synthase

tSpm

thermospermine

Supplementary Material

Supplementary Material
psb0602_0243SD1.pdf (1,023.8KB, pdf)

References

  • 1.Mahajan S, Tuteja N. Cold, salinity and drought stresses: An overview. Arch Biochem Biophys. 2005;444:139–158. doi: 10.1016/j.abb.2005.10.018. [DOI] [PubMed] [Google Scholar]
  • 2.Yamaguchi-Shinozaki K, Shinozaki K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Ann Rev Plant Biol. 2006;57:781–803. doi: 10.1146/annurev.arplant.57.032905.105444. [DOI] [PubMed] [Google Scholar]
  • 3.Tuteja N. Abscisic Acid and abiotic stress signaling. Plant Signal Behav. 2007;2:135–138. doi: 10.4161/psb.2.3.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Alcázar R, Marco F, Cuevas JC, Patron M, Ferrando A, Carrasco P, et al. Involvement of polyamines in plant response to abiotic stress. Biotechnol Lett. 2006;28:1867–1876. doi: 10.1007/s10529-006-9179-3. [DOI] [PubMed] [Google Scholar]
  • 5.Bartels D, Sunkar R. Drought and salt tolerance in plants. Crit Rev Plant Sci. 2005;24:23–58. [Google Scholar]
  • 6.Urano K, Maruyama K, Ogata Y, Morishita Y, Takeda M, Sakurai N, et al. Characterization of the ABA-regulated global responses to dehydration in Arabidopsis by metabolomics. Plant J. 2009;57:1065–1078. doi: 10.1111/j.1365-313X.2008.03748.x. [DOI] [PubMed] [Google Scholar]
  • 7.Alcázar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, et al. Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta. 2010;231:1237–1249. doi: 10.1007/s00425-010-1130-0. [DOI] [PubMed] [Google Scholar]
  • 8.Bouchereau A, Aziz A, Larher F, Martin-Tanguy J. Polyamines and environmental challenges: recent development. Plant Sci. 1999;140:103–125. [Google Scholar]
  • 9.Groppa MD, Benavides MP. Polyamines and abiotic stress: Recent advances. Amino Acids. 2008;34:35–45. doi: 10.1007/s00726-007-0501-8. [DOI] [PubMed] [Google Scholar]
  • 10.Gill SS, Tuteja N. Polyamines and abiotic stress tolerance in plants. Plant Signal Behav. 2010;5:26–33. doi: 10.4161/psb.5.1.10291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Kusano T, Berberich T, Tateda C, Takahashi Y. Polyamines: essential factors for growth and survival. Planta. 2008;228:367–381. doi: 10.1007/s00425-008-0772-7. [DOI] [PubMed] [Google Scholar]
  • 12.Janowitz T, Kneifel H, Piotrowski M. Identification and characterization of plant agmatine iminohydrolase, the last missing link in polyamine biosynthesis of plants. FEBS Lett. 2003;544:258–261. doi: 10.1016/s0014-5793(03)00515-5. [DOI] [PubMed] [Google Scholar]
  • 13.Piotrowski M, Janowitz T, Kneifel H. Plant C-N hydrolases and the identification of a plant N-carbamoylputrescine amidohydrolase involved in polyamine biosynthesis. J Biol Chem. 2003;278:1708–1712. doi: 10.1074/jbc.M205699200. [DOI] [PubMed] [Google Scholar]
  • 14.Hanfrey C, Sommer S, Mayer MJ, Burtin D, Michael AJ. Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity. Plant J. 2001;27:551–560. doi: 10.1046/j.1365-313x.2001.01100.x. [DOI] [PubMed] [Google Scholar]
  • 15.Slocum RD, Kaursawhney R, Galston AW. The physiology and biochemistry of polyamines in plants. Arch Biochem Biophys. 1984;235:283–303. doi: 10.1016/0003-9861(84)90201-7. [DOI] [PubMed] [Google Scholar]
  • 16.Panicot M, Minguet EG, Ferrando A, Alcázar R, Blazquez MA, Carbonell J, et al. A polyamine metabolon involving aminopropyl transferase complexes in Arabidopsis. Plant Cell. 2002;14:2539–2551. doi: 10.1105/tpc.004077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Urano K, Yoshiba Y, Najo T, Igarashi Y, Sek M, Sekiguchi F, et al. Characterization of Arabidopsis genes involved in biosynthesis of polyamines in abiotic stress responses and developmental stages. Plant Cell Environ. 2003;26:1917–1926. [Google Scholar]
  • 18.Cona A, Rea G, Angelini R, Federico R, Tavladoraki P. Functions of amine oxidases in plant development and defence. Trends Plant Sci. 2006;11:80–88. doi: 10.1016/j.tplants.2005.12.009. [DOI] [PubMed] [Google Scholar]
  • 19.Moller SG, McPherson MJ. Developmental expression and biochemical analysis of the Arabidopsis ATAO1 gene encoding an H2O2-enerating diamine oxidase. Plant J. 1998;13:781–791. doi: 10.1046/j.1365-313x.1998.00080.x. [DOI] [PubMed] [Google Scholar]
  • 20.Moschou PN, Sanmartin M, Andriopoulou AH, Rojo E, Sanchez-Serrano JJ, Roubelakis-Angelakis KA. Bridging the gap between plant and mammalian polyamine catabolism: A novel peroxisomal polyamine oxidase responsible for a full back-conversion pathway in Arabidopsis. Plant Physiol. 2008;147:1845–1857. doi: 10.1104/pp.108.123802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Moschou PN, Paschalidis KA. Roubelakis-Angelakis KA, Plant polyamine catabolism: The state of the art. Plant Signal Behav. 2008;3:1061–1066. doi: 10.4161/psb.3.12.7172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Takahashi Y, Cong R, Sagor GH, Niitsu M, Berberich T, Kusano T. Characterization of five polyamine oxidase isoforms in Arabidopsis thaliana. Plant Cell Rep. 2010;29:955–965. doi: 10.1007/s00299-010-0881-1. [DOI] [PubMed] [Google Scholar]
  • 23.Bartels D. Desiccation tolerance studied in the resurrection plant Craterostigma plantagineum. Integr Comp Biol. 2005;45:696–701. doi: 10.1093/icb/45.5.696. [DOI] [PubMed] [Google Scholar]
  • 24.Furini A. CDT retroelement: The stratagem to survive extreme vegetative dehydration. Plant Signal Behav. 2008;3:1129–1131. doi: 10.4161/psb.3.12.7076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Shinozaki K, Yamaguchi-Shinozaki K. Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol. 2000;3:217–223. [PubMed] [Google Scholar]
  • 26.Kakehi JI, Kuwashiro Y, Niitsu M, Takahashi T. Thermospermine is required for stem elongation in Arabidopsis thaliana. Plant Cell Physiol. 2008;49:1342–1349. doi: 10.1093/pcp/pcn109. [DOI] [PubMed] [Google Scholar]
  • 27.Knott JM, Romer P, Sumper M. Putative spermine synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine. FEBS Lett. 2007;581:3081–3086. doi: 10.1016/j.febslet.2007.05.074. [DOI] [PubMed] [Google Scholar]
  • 28.Alcázar R, Cuevas JC, Patrón M, Altabella T, Tiburcio AF. Abscisic acid modulates polyamine metabolism under water stress in Arabidopsis thaliana. Physiol Plant. 2006;128:448–455. [Google Scholar]
  • 29.Cuevas JC, López-Cobollo R, Alcázar R, Zarza X, Koncz C, Altabella T, et al. Putrescine is involved in Arabidopsis freezing tolerance and cold acclimation by regulating abscisic acid levels in response to low temperature. Plant Physiol. 2008;148:1094–1105. doi: 10.1104/pp.108.122945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Ríos G, Lossow A, Hertel B, Breuer F, Schaefer S, Broich M, et al. Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes. Plant J. 2002;32:243–253. doi: 10.1046/j.1365-313x.2002.01416.x. [DOI] [PubMed] [Google Scholar]
  • 31.Sasaki K, Abid MR, Miyazaki M. Deoxyhypusine synthase gene is essential for cell viability in the yeast Saccharomyces cerevisiae. FEBS Lett. 1996;384:151–154. doi: 10.1016/0014-5793(96)00310-9. [DOI] [PubMed] [Google Scholar]
  • 32.Chen KY, Liu AYC. Biochemistry and function of hypusine formation on eukaryotic initiation factor 5A. Biol Signals. 1997;6:105–109. doi: 10.1159/000109115. [DOI] [PubMed] [Google Scholar]
  • 33.Park MH, Lee YB, Joe YA. Hypusine is essential for eukaryotic cell proliferation. Biol Signals. 1997;6:115–123. doi: 10.1159/000109117. [DOI] [PubMed] [Google Scholar]
  • 34.Yamasaki H, Cohen MF. NO signal at the crossroads: polyamine-induced nitric oxide synthesis in plants? Trends Plant Sci. 2006;11:522–524. doi: 10.1016/j.tplants.2006.09.009. [DOI] [PubMed] [Google Scholar]
  • 35.Kuppusamy K, Walcher C, Nemhauser J. Cross-regulatory mechanisms in hormone signaling. Plant Mol Biol. 2009;69:375–381. doi: 10.1007/s11103-008-9389-2. [DOI] [PubMed] [Google Scholar]
  • 36.Neill S, Barros R, Bright J, Desikan R, Hancock J, Harrison J, et al. Nitric oxide, stomatal closure and abiotic stress. J Exp Bot. 2008;59:165–176. doi: 10.1093/jxb/erm293. [DOI] [PubMed] [Google Scholar]
  • 37.An ZF, Jing W, Liu YL, Zhang WH. Hydrogen peroxide generated by copper amine oxidase is involved in abscisic acid-induced stomatal closure in Vicia faba. J Exp Bot. 2008;59:815–825. doi: 10.1093/jxb/erm370. [DOI] [PubMed] [Google Scholar]
  • 38.Tun NN, Santa-Catarina C, Begum T, Silveira V, Handro W, Floh EI, et al. Polyamines induce rapid biosynthesis of nitric oxide (NO) in Arabidopsis thaliana seedlings. Plant Cell Physiol. 2006;47:346–354. doi: 10.1093/pcp/pci252. [DOI] [PubMed] [Google Scholar]
  • 39.Liu K, Fu H, Bei Q, Luan S. Inward potassium channel in guard cells as a target for polyamine regulation of stomatal movements. Plant Physiol. 2000;124:1315–1326. doi: 10.1104/pp.124.3.1315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem. 2010;48:909–930. doi: 10.1016/j.plaphy.2010.08.016. [DOI] [PubMed] [Google Scholar]
  • 41.Liu HP, Dong BH, Zhang YY, Liu ZP, Liu YL. Relationship between osmotic stress and the levels of free, conjugated and bound polyamines in leaves of wheat seedlings. Plant Sci. 2004;166:1261–1267. [Google Scholar]
  • 42.Zhou Q, Yu BJ. Changes in content of free, conjugated and bound polyamines and osmotic adjustment in adaptation of vetiver grass to water deficit. Plant Physiol Biochem. 2010;48:417–425. doi: 10.1016/j.plaphy.2010.03.003. [DOI] [PubMed] [Google Scholar]
  • 43.Toumi I, Moschou PN, Paschalidis KA, Bouamama B, Ben Salem-Fnayou A, Ghorbel AW, et al. Abscisic acid signals reorientation of polyamine metabolism to orchestrate stress responses via the polyamine exodus pathway in grapevine. J Plant Physiol. 2010;167:519–525. doi: 10.1016/j.jplph.2009.10.022. [DOI] [PubMed] [Google Scholar]
  • 44.Bae H, Kim SH, Kim MS, Sicher RC, Lary D, Strem MD, et al. The drought response of Theobroma cacao (cacao) and the regulation of genes involved in polyamine biosynthesis by drought and other stresses. Plant Physiol Biochem. 2008;46:174–188. doi: 10.1016/j.plaphy.2007.10.014. [DOI] [PubMed] [Google Scholar]
  • 45.Yang JC, Zhang JH, Liu K, Wang ZQ, Liu LJ. Involvement of polyamines in the drought resistance of rice. J Exp Bot. 2007;58:1545–1555. doi: 10.1093/jxb/erm032. [DOI] [PubMed] [Google Scholar]
  • 46.Peremarti A, Bassie L, Christou P, Capell T. Spermine facilitates recovery from drought but does not confer drought tolerance in transgenic rice plants expressing Datura stramonium S-denosylmethionine decarboxylase. Plant Mol Biol. 2009;70:253–264. doi: 10.1007/s11103-009-9470-5. [DOI] [PubMed] [Google Scholar]
  • 47.Capell T, Bassie L, Christou P. Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress. Proc Natl Acad Sci USA. 2004;101:9909–9914. doi: 10.1073/pnas.0306974101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Wen XP, Ban Y, Inoue H, Matsuda N, Moriguchi T. Aluminum tolerance in a spermidine synthase-overexpressing transgenic European pear is correlated with the enhanced level of spermidine via alleviating oxidative status. Environ Exp Bot. 2009;66:471–478. [Google Scholar]
  • 49.Alcázar R, Planas J, Saxena T, Zarza X, Bortolotti C, Cuevas J, et al. Putrescine accumulation confers drought tolerance in transgenic Arabidopsis plants overexpressing the homologous Arginine decarboxylase 2 gene. Plant Physiol Biochem. 2010;48:547–552. doi: 10.1016/j.plaphy.2010.02.002. [DOI] [PubMed] [Google Scholar]
  • 50.Kusano T, Yamaguchi K, Berberich T, Takahashi Y. The polyamine spermine rescues Arabidopsis from salinity and drought stresses. Plant Signal Behav. 2007;2:251–252. doi: 10.4161/psb.2.4.3866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Yamaguchi K, Takahashi Y, Berberich T, Imai A, Takahashi T, Michael AJ, et al. A protective role for the polyamine spermine against drought stress in Arabidopsis. Biochem Biophys Res Commun. 2007;352:486–490. doi: 10.1016/j.bbrc.2006.11.041. [DOI] [PubMed] [Google Scholar]
  • 52.Kalamaki MS, Merkouropoulos G, Kanellis AK. Can ornithine accumulation modulate abiotic stress tolerance in Arabidopsis? Plant Signal Behav. 2009;4:1099–1101. doi: 10.4161/psb.4.11.9873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Mattoo AK, Minocha SC, Minocha R, Handa AK. Polyamines and cellular metabolism in plants: transgenic approaches reveal different responses to diamine putrescine versus higher polyamines spermidine and spermine. Amino Acids. 2010;38:405–413. doi: 10.1007/s00726-009-0399-4. [DOI] [PubMed] [Google Scholar]
  • 54.Mehta RA, Cassol T, Li N, Ali N, Handa AK, Mattoo AK. Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality and vine life. Nat Biotechnol. 2002;20:613–618. doi: 10.1038/nbt0602-613. [DOI] [PubMed] [Google Scholar]
  • 55.Grienenberger E, Besseau S, Geoffroy P, Debayle D, Heintz D, Lapierre C, et al. A BAHD acyltransferase is expressed in the tapetum of Arabidopsis anthers and is involved in the synthesis of hydroxycinnamoyl spermidines. Plant J. 2009;58:246–259. doi: 10.1111/j.1365-313X.2008.03773.x. [DOI] [PubMed] [Google Scholar]
  • 56.Bartels D, Schneider K, Terstappen G, Piatkowski D, Salamini F. Molecular cloning of abscisic acid modulated genes which are induced during desiccation of the resurrection plant Craterostigma plantagineum. Planta. 1990;181:27–34. doi: 10.1007/BF00202321. [DOI] [PubMed] [Google Scholar]
  • 57.Marce M, Brown DS, Capell T, Figueras X, Tiburcio AF. Rapid High-Performance Liquid-Chromatographic method for the quantitation of polyamines as their dansyl derivatives—application to plant and animal-tissues. J Chromatogr B. 1995;666:329–335. doi: 10.1016/0378-4347(94)00586-t. [DOI] [PubMed] [Google Scholar]
  • 58.Alcázar R, García-Martínez JL, Cuevas JC, Tiburcio AF, Altabella T. Overexpression of ADC2 in Arabidopsis induces dwarfism and late-flowering through GA deficiency. Plant J. 2005;43:425–436. doi: 10.1111/j.1365-313X.2005.02465.x. [DOI] [PubMed] [Google Scholar]
  • 59.Wi SJ, Kim WT, Park KY. Overexpression of carnation S-adenosylmethionine decarboxylase gene generates a broad-spectrum tolerance to abiotic stresses in transgenic tobacco plants. Plant Cell Rep. 2006;25:1111–1121. doi: 10.1007/s00299-006-0160-3. [DOI] [PubMed] [Google Scholar]
  • 60.Okuyama T, Kobayashi Y. Determination of Diamine Oxidase activity by liquid scintillation counting. Arch Biochem Biophys. 1961;95:242. doi: 10.1016/0003-9861(61)90141-2. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

Supplementary Material
psb0602_0243SD1.pdf (1,023.8KB, pdf)

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