Polyamine and Ethylene pathways crosstalk revisited on a genome wide and gene function scale

Polyamine and ethylene biosynthesis pathway genes are widely involved in the regulation of plant abiotic stresses. For their biosynthesis, both pathways require the same precursor, Synthase Adenosyl Methionine (SAM) enzyme. Whether they function as competitors or collaborators to regulate plant abiotic stress tolerance is still an elusive topic. Genome wide analysis of Cleistogenes songorica polyamine and ethylene pathway gene families was conducted to study their evolutionary relationship. And, using Arabidopsis plants transformed with a polyamine gene SAMDC2 from C. songorica, the expression of key genes from both pathways, and other previously well-studied stress responsive genes was investigated under salt or drought stress. Further, the ABA’s role on this interaction salt stress was also studied. 17 polyamine, 12 ethylene and 6 SAM biosynthesis related genes were identified at genome wide level in C. songorica. Phylogenetic analysis revealed close evolutionary similarities between gene families from both pathways. Also, analysis of cis regulatory elements indicated that SAM family genes promoters were rich into both ABA and ethylene related cis regulatory elements. Transcriptomic analysis, qRT-PCR validation, and confirmation using transgenic Arabidopsis showed that polyamine and ethylene key pathway genes can be concurrently expressed during abiotic stresses. Arabidopsis plants expressing a polyamine gene CsSAMDC2 driven by RD29A showed an improved drought and salt stress tolerance, and an increased expression of key polyamine and ethylene pathway genes. These plants maintained higher chlorophyll content and photosynthetic capacity. Morphological analysis of transgenic seedlings showed that leaves of these lines exhibited a more compact architecture following salt stress exposure. In silico and gene functional analysis assays revealed potential evolutionary and functional similarities between polyamine and ethylene pathway gene families. Such findings imply a synergetic interaction between polyamine and ethylene pathways, and the significant role of ABA on this crosstalk. Abstract Background: Polyamine and ethylene biosynthesis pathway genes are widely involved in the regulation of plant abiotic stresses. For their biosynthesis, both pathways require the same precursor, Synthase Adenosyl Methionine(SAM) enzyme.Whether they function as competitors or collaborators to regulate plant abiotic stress tolerance is still an elusive topic. Genome wide analysis of Cleistogenes songorica polyamine and ethylene pathway gene families was conducted to study their evolutionary relationship. And, using Arabidopsis plants transformed with a polyamine gene SAMDC2 from C. songorica , the expression of key genes from both pathways, and other previously well-studied stress responsive genes was investigated under salt or drought stress. Further, the ABA’s role on this interaction salt stress was also studied. Results: 17 polyamine, 12 ethylene and 6 SAM biosynthesis related genes were identified at genome wide level in C. songorica . Phylogenetic analysis revealed close evolutionary similarities between gene families from both pathways. Also, analysis of cis regulatory elements indicated that SAM family genes promoters were rich into both ABA and ethylene related cis regulatory elements. Transcriptomic analysis, qRT-PCR validation, and confirmation using transgenic Arabidopsis showed that polyamine and ethylene key pathway genes can be concurrently expressed during abiotic stresses. Arabidopsis plants expressing a polyamine gene CsSAMDC2 driven by RD29A showed an improved drought and salt stress tolerance, and an increased expression of key polyamine and ethylene pathway genes. These plants maintained higher chlorophyll content and photosynthetic capacity. Morphological analysis of transgenic seedlings showed that leaves of these lines exhibited a more compact architecture following salt stress exposure. during salt stress and ABA treatments. Interestingly, the expression of ethylene pathway genes was not reversed by exogenous ABA during salt stress treatment. Conclusion: In silico and gene functional analysis assays revealed potential evolutionary and functional similarities between polyamine and ethylene pathway gene families. Such findings imply a synergetic interaction between polyamine and ethylene pathways, and the significant role of ABA on this crosstalk. CGTCA-motif

polyamine and ethylene pathway protein sequences were aligned with Arabidopsis and O. sativa polyamine and ethylene protein sequences. All the protein sequences with their respective accession numbers are shown in Additional file 6. The multiple proteins sequence alignment was performed by using ClustalW algorithm (Feng and Doolittle 1987) within MEGA 6 software. The resulting protein sequence alignments were then used to construct phylogenetic tree after pair-wise deletion of gaps with p-distance matrix [36]. The Neighbor-Joining algorithm was used with one thousand bootstrap [36,37].
cis-regulatory element and motif analysis C. songorica sequences of 2000 bp of the gene promoter region corresponding with the identified polyamine and ethylene genes were analyzed for putative cis-regulatory elements using PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [38]. We then recorded and compared the number of stress and hormonal-related cis-regulatory elements in individual genes from polyamine and ethylene pathway, and from SAM family.
Transcriptomic analyses and qRT-PCR validation C. songorica seeds were sown in vermiculite medium supplied with 1/4 diluted Hoagland's nutrient solution, pH 5.8. Growth chamber conditions were set at 75-80 % relative humidity, 30 / 28°C (day / night), and 16 /8 h (day / night) at the light intensity of 200 mmol photons m −2 s −1 . For heat stress treatment, one-month old seedlings were treated at 40 0 C for 24 h. Dehydration stress treatment was done by withholding irrigation to 6-10% of soil water content (for mild drought stress), and to 1-3% for severe dehydration stress. For salt stress treatment, different NaCl concentrations (50mM; 100mM; 200 mM) were prepared and applied on seedlings by spraying. In another set of experiment seedlings were treated with 100 μM ABA to investigate the role of ABA on polyamine and ethylene pathway genes expression. For each treatment, root and shoot tissue samples were collected at 0 and 24 h post treatment and kept at −80 °C till RNA extraction. Three samples were collected for each treatment. Total RNA was isolated from root or shoot samples using the Shengong RNA isolation kit as instructed (Shengong Ltd, Shanghai, China). The isolated RNA samples were further pooled, and sequenced following Illumina sequencing guidelines. The root and shoot total RNA samples were also used to synthesize first strand cDNAs using an oligo dT primer and the cDNA synthesis kit (Shengong). The cDNAs samples were individually diluted to 100 ng/μL prior to qPCR using gene specific primers (Additional file 2). For each PCR reaction, three biological replicates with three technical replicates each were used. qPCR reaction was conducted using 40 cycles of 95 °C 5 s, 60 °C 15 s, and 72 °C 34 s, a SYBR Green Master (Shengong), and gene specific primers. The relative gene expression levels were analyzed using the comparative ΔΔCt method [39]. The expression level of C. songorica GADPH gene was used as an internal control. Two ways analysis of variance and Duncan's multiple range test (DMRT) were used for multiple mean comparisons. SPSS (IBM Corp. 2013, IBM SPSS Statistics for Windows, Version 21.0, Armonk, NY) was used to determine the significant differences between means (p< 0.005).
Cloning and transformation of a polyamine gene, CsSAMDC2 into Arabidopsis The amplification of cDNA CsSAMDC2 (EST) was performed using a SMARTTM RACE cDNA Amplification Kit (Clontech, Japan). After sequencing, both 5' and 3' ends primers (Supplementary   (Table 1). Strikingly, the average number of MYB and ABRE cis-regulatory elements was almost comparable in both polyamine and ethylene pathway gene promoter regions, with a slight difference in favor of polyamine genes. In average, 1.3 ABRE and 2 MYB cis acting elements were counted in the promoter regions of polyamine pathway genes, while 1 ABRE and 1.4 MYB were recorded for ethylene pathway genes. Promoters of SAM family genes were the most enriched in ABRE cis regulatory elements and averaged of 3.1 counts per gene. Also, higher number of G-box, W-box and Jasmonic acid inducible motifs, CGTCA-motif and TGACG-motif was recorded in the promoter regions of SAM family genes.

Transcriptomic analysis
Expression analysis revealed that many of SAM family genes were constitutively expressed across normal irrigations, mild drought stress and high dehydration stress treatments ( Fig. 2A). CsSAM1 and CsSAM5 gene expression levels were induced after dehydration treatments in both root and shoot tissues, and consequently their expression was reduced after re-watering. The expression level of CsSAM6 gene was consistently high across all the treatments, and re-watering remarkably reduced its expression particularly in root. Expression levels of polyamine and ethylene pathway genes were also analyzed under different levels of salt stress, heat stress, low temperature stress, and under ABA treatment (Fig. 2B). For ethylene pathway, the expression level of Cs2OG(FeII)1 gene increased after applying 200 mM NaCl stress conditions, interestingly, ABA treatment did not reverse its expression in shoot. Similarly, the expression levels of Cs2OG(FeII)3 gene increased in both tissues following 100 mM NaCl, heat and ABA treatments. Also, the expression levels of another ethylene pathway gene CsACC1 were induced by 50 mM of NaCl and low temperature treatment in shoot and root tissues, and its expression went up in root after ABA treatment. In the same metabolic pathway, CsACC3 gene exhibited an increased expression in both tissues after 100 mM NaCl treatment, and its expression was also high in root after normal treatment conditions. For polyamine pathway genes, the relative expression level of CsADC2 gene was slightly induced by 100 mM NaCl stress in roots and by ABA treatment in shoots. For SAMDC family genes, CsSAMDC4 gene expression was induced by all the treatments in shoot tissue. CsSPMS genes were constitutively expressed across all treatments in both tissues, and ABA slightly increased the expression levels of CsSPMS1, CsSPMS3 and CsSPMS4. The expression level of CsSPMS2 was highly induced in roots after 100 mM NaCl or 200 mM NaCl and heat treatments in both tissues. On the other hand, expression levels of SAM family genes were generally higher compared to other families in both pathways, and from all the analyzed genes, CsSAM6 gene exhibited the highest expression level in shoot tissues after ABA treatment (Fig. 2B) qRT-PCR validation Quantitative RT-PCR validations of these expression profiles were conducted for drought, salt stress and ABA treatments in root and shoot tissues (Fig. 3). For genes involved in ethylene biosynthesis pathway, CsACC3 was selected as a representative (Fig. 3A). qRT-PCR validated the expression of CsACC3 as it was induced by 16.87 times in shoot after strong dehydration stress, while its expression in root increased from 2.7 folds after 50 mM NaCl to 5.4 folds under 100 Mm NaCl treatment, and then dropped to 2.6 folds at 200 mM NaCl. Treatment with ABA did not reduce its expression levels relative to control conditions. This trend correlates well with the findings from the transcriptomic analysis.
The expression of CsSAM6 gene, was induced by all levels of drought and salt treatment applied, more importantly, as observed during transcriptome analysis, its expression level was the highest in shoot after ABA treatment as it soared by 18.3 folds (Fig. 3B). The expression CsSAMDC2 was induced by all stress treatments and ABA application. The expression level of CsSAMDC2 gene after strong dehydration stress in shoot was the highest as it increased by 754 times relative to control. Also, its expression was induced to 11.7 and 16.4 times in root and shoot tissues following ABA treatment (Fig.   3C).
Expression of CsSAMDC2 gene enhanced drought stress tolerance in transgenic Arabidopsis As CsSAMDC2 gene was highly induced in C. songorica under drought conditions, it was transformed into Arabidopsis under a stress responsive and constitutive promoter, RD29A and 35S, respectively.
Using primers of CsSAMDC2 gene promoters, and transformed Arabidopsis genomic DNA, an amplicon band of around 1200 bp size was detected in 9 Arabidopsis lines (Additional file 1). Similarly, successful transformation of RD 29A and 35S promoters was also confirmed through PCR using Wild-type Arabidopsis and transgenic lines expressing CsSAMDC2 gene were grown on 150 mM NaCl stress medium. After reaching maximum germination rate (after 28 days) (Additional file 4A), all seedlings were maintained under the same treatment conditions for18 days (Fig. 6A). The number of cotyledons that maintained green colors after treatment was found higher in transgenic seedlings than in their wild-type plants (Fig. 6B). Moreover, leaves of transgenic Arabidopsis showed a more compact architecture (Fig. 6A), a typical salt avoidance mechanism in stress tolerant plants [46,47].

Expression of Polyamine and Ethylene pathway genes and other stress responsive genes under different treatments
The expression of different stress responsive genes including polyamine related genes AtSAMDC2 and AtADC2, and ethylene pathway genes, AtACO2 and AtACS6, an Ethylene responsive gene AtERF1, and ABA responsive gene AtRD29A were more improved in transgenic Arabidopsis after drought stress treatment than in their wild-type counterparts (Fig. 7A).
All studied polyamine and ethylene pathway genes showed positive upregulations after salt stress ( Fig. 7B) or salt stress +ABA treatments (Fig. 7C). Analysis of the expression of key polyamine pathway genes AtSAMDC2 and AtADC2 under salt stress +ABA treatments revealed that their expression levels increased by 24 (AtSAMDC2) and 21 (AtADC2) folds in TL01, and 29 (AtSAMDC2) and 28.7(AtADC2) folds in TL18. (Fig. 7C). However, the expression of both genes only increased by 4.9 (AtADC2) and 4.7 folds (AtSAMDC2), respectively, in wild-type plants relative to non-treatment conditions. The activity of AtRD29A was also highly induced by salt stress (Fig. 7B), and ABA application enhanced its expression levels to 89 and 87 folds in TL01 and TL018 plants (Fig. 7C) respectively, which were the highest recorded expression levels in all lines analyzed.
Exogenous ABA altered plant architecture of transgenic Arabidopsis under salt tolerance Analysis of leaves and root morphology in wild-type and transgenic lines grown on MS plates supplemented with 150 mM NaCl indicated no significant phenotypical differences between these lines after 72 h (Fig. 8B). However, application of ABA induced formation of lateral roots and triggered root growth in all lines compared to salt stress treatment alone (Fig. 8D). RD29A::CsSAMDC2 lines exhibited the most improved lateral root development under these treatments conditions (Fig. 8D). The present study suggests that under stress conditions both pathways may enter a mutually beneficial synergistic relationship instead of merely competing for SAM. And for the first time, this hypothesis was studied on genome wide scale. Results from phylogenetic analysis indicated close evolutionary relationships between some gene families from polyamine and ethylene pathways.

Measurements of root length in
These findings may infer functional similarities that could have narrowed divergence of these families during their evolutionary history. A such gene functional evolution concept corroborates with former studies in Brassica napus LEA gene family [52]. Also, SAM genes were found to be rich into cis acting elements which provide binding sites for both ethylene, Jasmonic and ABA related transcription factors. This could explain their functional relevance in mediating both polyamine and ethylene pathways during stresses conditions. The above results were further supported by a simultaneous increased expression of both polyamine and ethylene pathway genes, ethylene and ABA responsive gene in Arabidopsis expressing a polyamine gene CsSAMD2 during salt or drought stresses.
Other earlier studies suggested that instead of metabolically competing for SAM, other molecular mechanisms may play into their role [53,54]. For instance, it was proposed that the activity of AtERF1 gene enhanced plant tolerance to a broad spectrum of abiotic stresses by integrating Abscisic acid, ethylene and Jasmonic acid [55]. To investigate this hypothesis, exogenous ABA was applied on salt-stressed transgenic lines and wild type plants. Results showed that during salt stress, application of ABA further enhanced mRNA expression levels for polyamine (AtSAMDC2 and AtADC2) and ethylene pathway genes (AtACO2 and AtACS6), and for ABA (AtRD29A) and ethylene (AtERF1) responsive genes in transgenic Arabidopsis under salt stress. Interestingly, the expression of AtERF1 was found higher in RD29A::CsSAMDC2 lines than in 35S::CsSAMDC2 andwild-type plants.
Furthermore, ABA predominantly promoted the expression of AtRD29A and AtSAMDC2 in RD29A::CsSAMDC2 Arabidopsis lines; TL01 and TL018. Subsequently, TL01 and TL018 showed improved primary and lateral and root growth. Previous studies showed that ABA induces lateral roots development under salt stress [56,57] through its signaling that are transduced in cell specific manner and through intricate hormonal interactions [58,59]. However, some previous studies reported that lateral and primary root growth suppress each other's development [58]. Interestingly, in this study both traits were found enhanced in Arabidopsis plants expressing RD29A::CsSAMDC2 gene construct. This was possibly due to an enhanced activity polyamines genes, AtADC2 and AtSAMDC2. Polyamines are known to promote cell division and differentiation necessary for root apex and lateral root development [60]. It was previously shown that disrupting the activity of ADC genes in Arabidopsis [61] or in P. vulgaris [62] correlated with reduced lateral root formation and primary root growth, respectively. Similarly, downregulating SAMDC gene in potato plants was associated with limited root growth [63].
This study showed that though CsSAMDC2 may suppress salt stress under ABA independent manner, exogenous ABA could promote its activity by enhancing the upregulation of polyamine (AtADC2 and AtSAMDC2), ethylene (AtERF1, AtACO2, AtACO6) and ABA (AtRD29A) related stress responsive genes.
From these findings we speculate that under abiotic stress ABA, ethylene and polyamine could mutually interact to respond to their respective functions in plant defensive mechanism. Moreover, as a results of a rich diversity cis acting elements found in the promoter regions of SAM family genes, we speculate that SAM enzymes may function as mediator for polyamine and ethylene pathways during stress conditions. A network for stress response regulation by polyamine, ethylene pathway genes through SAM was proposed in this study (Fig. 9), however further molecular assays are required for confirmation.

Conclusions
Phylogenetic and cis-regulatory analyses revealed potential evolutionary and functional similarities between polyamine and ethylene pathway gene families. Subsequently, the expression of genes from both pathways was induced during drought or salt stresses, or salt stress +ABA treatments in Arabidopsis expression a polyamine gene, CsSAMDC2. Findings from this study suggest molecular interaction between polyamine and ethylene pathways genes during salt or drought stresses, and the important role of ABA on this crosstalk during salt stress.