CHIRALITY (2012)

Selective Regulation of Nicotine and Polyamines Biosynthesis inTobacco Cells by Enantiomers of Ornithine

MORTEZA GHOLAMI,1 ALIREZA FAKHARI,1* AND FAEZEH GHANATI2*1Medicinal Plants and Drugs Research Institute (MPDRI), Shahid Beheshti University, G.C., Tehran, Iran

2Department of Plant Biology, Faculty of Biological Science, Tarbiat Modares University (TMU), Tehran, Iran

© 2012 Wiley Perio

ABSTRACT L- and D-amino acids have diverse functions and effects on the metabolism,growth, and development of plants. Ornithine (Orn) plays a main role in the biosynthesis ofmany amino acids, nicotinic alkaloids, and polyamines in tobacco. This investigation describesthe impact of Orn enantiomers on the production and distribution of free, conjugated, andbound polyamines, as well as nicotine in tobacco cells. It was recognized that the biosynthesisof metabolites was differently upregulated by each enantiomer. Putrescine was abundantlyproduced by exogenous L-ornithine (L-Orn), and both spermidine and spermine weresignificantly accumulated in D-ornithine (D-Orn)-supplied tobacco cells. Furthermore, nicotineproduction was highly upregulated by L-Orn, while the addition of D-Orn had no effect on thenicotine content of tobacco cells. It was observed that transcript expression of S-adenosylmethioninedecarboxylase, as the key enzyme of spermidine/spermine biosynthesis, is coincident with theirmetabolic levels and is highly upregulated by D-Orn, as opposed to L-Orn. These results indicatethat both enantiomers of Orn can trigger selected biosynthetic pathways in the cells, at thetranscript level. Regarding these observations, it is proposed that L- and D-Orn function differentlyin the same biological pathways in which the latter, D-Orn specifically regulates importantpolyamines in the plant cells. Chirality 00:000–000, 2012. © 2012 Wiley Periodicals, Inc.

KEY WORDS: D-amino acids; (D/L)-ornithine; Nicotiana tabacum; nicotine; polyamines

*Correspondence to: Alireza Fakhari, Medicinal Plants and Drugs ResearchInstitute (MPDRI), Shahid Beheshti University, G.C., Tehran, Iran. E-mail:a-zavareh@sbu.ac.ir; Faezeh Ghanati, Department of Plant Biology, Facultyof Biological Science, Tarbiat Modares University (TMU), Tehran, Iran.E-mail: ghangia@modares.ac.irReceived for publication 6 May 2012; Accepted 12 July 2012DOI: 10.1002/chir.22107Published online in Wiley Online Library(wileyonlinelibrary.com).

INTRODUCTIONWhile L-amino acids (L-AAs) are thought to be the most

abundant enantiomers in nature, D-amino acids (D-AAs) arepresent in organisms in far greater quantities than previouslyconsidered. The physiological function and pathologicalimportance of D-AAs occurring principally in the nervousand endocrine systems have been extensively investigated,for example, D-serine has been found in the mammaliancentral nervous system, D-aspartate in mammalian neuroen-docrine organs, and D-alanine and D-aspartate in aquaticanimals.1 Although there is substantial information availableabout D-AAs in animals, there is limited information of D-AAsin plants and their potential role in metabolism, growth, anddevelopment. In plants, occurrence of D-AAs is derived frommicrobial contamination, racemization of L-AAs, or the functionof some biosynthetic enzymes. In comparison with bacteria,fungi, and animals, plants lack the enzymes for metabolizingD-AAs that results in the inhibitory effects of certain D-AAs inplants growth and development.2–4 Investigations on aminoacid transporters in plants have shown that D-enantiomersof amino acids are transported at lower rates, compared withL-enantiomers.5 It is also shown that profile of other AAs andN metabolism is different depending on AAs enantiomers.6,7

L-Ornithine (L-Orn) is a non-protein amino acid that plays acentral role in the biosynthesis of nicotine, polyamines (PAs),and in the urea cycle.8 It plays a major role in signal transduc-tion as a sensory and regulatory molecule and is shown to beaccumulated under stress conditions in plant cells.9,10 As the firstPA, putrescine (Put) is biosynthesized from Orn by ornithinedecarboxylase (ODC), which is then further aminopropylatedby spermidine (Spd) and spermine (Spm) synthases (SPDSand SPMS, respectively) and S-adenosylmethionine decarboxyl-ase (SAMDC) to form Spd and Spm, respectively. Production of

dicals, Inc.

nicotine from Put by the action of putrescinemethyl transferaseis a competitor of Spd biosynthesis in tobacco (Fig. 1).Polyamines are present in all compartments of the plant

cells and are involved in the regulation of genome activity,cell division, expansion, plant growth, and development.11 Innature, PAs often occur as free molecular bases (free PAs)but can also be associated with small molecules like phenolicacids (perchloric acid soluble conjugated PAs) or macromole-cules like proteins (perchloric acid insoluble bound PAs).12

Free PAs have important antioxidant properties comparedwith conjugated polyamines that are important for the controlof the intracellular PAs concentrations and also for theirinteraction with components of the cell wall.13,14 Boundpolyamines have strong interactions with membrane proteins,DNA, RNA, and chromosomes and carry out a vital role inthe transcription process and nucleotide-related stressessuch as ultraviolet stress.15 Although the function of D-AAson plant growth and development is being extensivelystudied, understanding of their effects on the metabolitebiosynthesis of the plants is limited. This study was aimed toelucidate the effects of L- and D-Orn on the production ofrelated metabolites, in particular on the production of PAs, insuspension-cultured tobacco. It was of interest to observehow biosynthetic pathways are affected by change ofthe enantiomer of AA. This goal can be achieved either by

Fig. 1. Biosynthesis of polyamines and nicotine from ornithine in tobacco.ODC, ornithine decarboxylase; PMT, putrescinemethyl transferase; SPDS,spermidine synthesis; SAMDC, S-adenosylmethionine decarboxylase; SPMS,spermine synthesis.

GHOLAMI ET AL.

tracking isotope-labeled enantiomers or instead by monitor-ing the expression of key enzymes and their putative pro-ducts. Due to lack of isotope-labeled D-Orn, the expressionof a key enzyme (SAMDC) and production of free, conjugated,and bound PAs along with nicotine (products) were evaluated.

MATERIALS AND METHODSCell Culture and Treatments

Suspension-cultured tobacco (Nicotiana tabacum L. cv. Burley 21) cellswere grown in a modified Murashige and Skoog medium without glycine,containing 3% sucrose.16 The cells in their logarithmic growth phase (day5) were treated with or without 1, 5, and10mM of L- and D-Orn for 24 h.The cells were then harvested, washed thoroughly with deionized water,frozen in liquid N2, and kept at �80 �C for further analysis.

ChemicalsEnantiomers of Orn, high purity (99%), and PAs standards were pur-

chased from Sigma-Aldrich (St. Louis, MO, USA). All chromatographicsolvents used were HPLC grade, purchased from Caledon (Canada).

Extraction of Free, Conjugated, and Bound Polyamines andTheir Quantification

Free, conjugated, and bound PAs were quantified according to ourprevious report,17 with minor modifications. Samples of fresh cells(0.2 g) were homogenized in 1ml of 5% (v/v) perchloric acid (PCA) andincubated at 4 �C for 1 h. The homogenate was centrifuged at 12,000gfor 20min at 4 �C, and the supernatant and pellet were collected sepa-rately. The supernatant was used to determine the free and conjugatedPAs, whereas the pellet was used to measure PCA-insoluble bound PAsas follows. In order to extract the PCA-soluble conjugated PAs, 0.2ml ofthe supernatant was mixed with 0.2ml of 12M HCl and heated at110 �C for 18 h in flame-sealed glass ampoules. After acid hydrolysis,HCl was evaporated from the tubes by further heating at 80 �C, and theresidue was re-suspended in 0.5ml of 5% (v/v) PCA. This solution wasused as a source of PCA-soluble conjugated PAs. In order to extractPCA-insoluble bound PAs, the pellet was rinsed three times with 0.5mlof 5% PCA to remove any trace of soluble PAs and then centrifuged at12,000g for 20min, and the supernatant, including the bound PAs, washydrolyzed under the same conditions as mentioned for PCA-solubleconjugated PAs. For derivatization, an aliquot (200ml) of each extract

Chirality DOI 10.1002/chir

was added to 200ml of saturated Na2CO3, and 400ml of dansyl chloride(5mg in 1ml of acetone) at room temperature, mixed, and incubated at70 �C in the darkness for 90min. Excess dansyl chloride was inactivatedby adding 200ml of proline (100mg/ml) for 30min. Dansylated PAs wereextracted into 0.5ml of toluene after 1min of vortex stirring. The upperorganic phase was collected and stored at �20 �C for high performancethin layer chromatography system (HPTLC) analysis. Thin layerchromatography was performed using an HPTLC equipped with Linomat5 autosampler, TLC scanner 3, and winCATS 1.2.2 software (CAMAG,Muttens, Switzerland). Chromatography was performed on TLC precoatedsilicagel 60 F254 plate (10� 20 cm, Merck, Darmstadt, Germany) usingcyclohexane : toluene (5:4) as the mobile phase. Fluorescence measure-ment of compounds was performed via peak area after scanning withexcitation and emission at l = 250 and 366nm, respectively. Quantificationof PAs was performed with using a calibration curve obtained for each PAin the range of 0–1000ppm.

Determination of Nicotine by Gas Chromatography-MassSpectrometry (GC-MS)

A 10mg of dried tobacco cells was weighed into a tube with theaddition of 2-ml ether (containing 10.0 mg/ml n-heptadecane, as an inter-nal standard) and 1ml of 5% sodium hydroxide (NaOH) was added.18

After ultrasonication for 15min, the two layers were separated, and 1 mlof the sample from ether layer was injected into the gas chromatographto be analyzed. The GC-MS system used was a 7890A Agilent gaschromatograph and a 5975C Agilent quadrupole mass spectrometer(Agilent, Santa Clara, CA, USA). The column used was a 30-m VF-5MScolumnwith 0.2-mm film thickness.The analysis of samples was performedunder the following temperature program; start at injection 70 �C, a holdfor 1min, followed by a 7 �C/min oven temperature ramp to 250 �C,and a final 5-min heating at 250 �C. The column used was a 30-mVF-5MS column with 0.2-mm film thickness.

RNA Extraction and Semi Quantitative Reverse TranscriptasePolymerase Chain Reaction Analysis

The change of transcription level of the genes in different treatmentswas monitored by semi-quantitative RT-PCR technique. The expressionof the actin gene was used as an internal control. Total RNA was preparedfrom the cells using RNX™+ (CinnaGen Inc. Tehran, Iran), following themanufacturer’s instructions. The quality and quantity of RNA were exam-ined using agarose gel electrophoresis and spectroscopy, respectively.The purified RNA (3mg) was used for synthesis of first-strand cDNAusing oligo dT18 as a primer and first-strand cDNA synthesis Kit(Fermentas, Burlington, Ontario, Canada) according to the manufac-turer’s instruction. PCR was performed in 20-ml reaction mixture containing0.3ml of first-strand cDNA, 0.5ml of recombinant Taq DNA polymerase(CinnaGen), 10mM Tris-HCl (pH = 8.8), 50mM KCl, 1.5mM MgCl2,200 mM of each deoxynucleoside triphosphate, and 4 mM of forwardand reverse primers. The PCR amplification of genes transcripts wasperformed by initial denaturation at 94 �C for 1min, and the amplificationwas performed by 30 cycles of denaturation for 30 sec, annealingbetween 52 and 58 �C for 25 sec depending on the gene of study, andextension at 72 �C for 10min. PCR products were separated on 1.2%agarose gel electrophoresis. The band intensity on the gel stained withethidium bromide was measured by ultraviolet Documentation Lumi-nescent Image Analysis software (Raytek, Sheffield, England, UK).The nucleotide sequences of amplified fragment of genes were con-firmed from both strands sequencing (Macrogen, Korea) (AB536743.1).Semi-quantitative RT-PCR was performed using gene-specific primers

of ACT (forward: 50-GCAGGGATCCACGAGACCACC-30; reverse:50-CCCACCACTGAGCACAATGTTCC-30), and SAMDC from Nicotianatabacum (forward: 50- TTGGTAGCAACATCAGCATGCA-30; reverse:50-TGACCCTGTTTACACTCTTGAG-30).

Statistical AnalysisAll of the experiments were carried out with at least three independent

repetitions using three samples, and all of the data were expressed as themean values� the standard deviation. Statistical analysis was performed

EFFECT OF ORNITHINE ENANTIOMERS ON THE METABOLISM OF TOBACCO

using student’s t-test, and the differences between treatments wereexpressed as significant at level of P 0.05.

RESULTSQuantification of the results obtained from HPTLC showed

that both Orn enantiomers (D and L) increased the PAscontent in tobacco cells, and free Put was found to be slightlyhigher in L-Orn-treated cells, compared with those treatedwith D-Orn (Fig. 2). Furthermore, the level of free Spd andSpm was significantly higher in the D-Orn-treated cells. Thelevels of both Spd and Spm in D-Orn treatments (10mM)were up to twofolds than their level in the control condition.Since conjugated and bound PAs are produced from free

forms, it is important to measure the contents of conjugateand bound PAs as well. As illustrated in Figure 3, treatmentof tobacco cells with L-Orn resulted in higher contents ofconjugated Put than that of D-Orn-treated cells. ConjugatedSpd and Spm, however, were fairly higher in D-Orn-treatedcells (Fig. 3).Bound Spm content was below the detection limits and was

not detected in L- or D-Orn-treated cells. Bound Put increasedwhen the cells were treated with L-Orn, whereas D-Orn-treated cells produced higher amounts of bound Spd, inagreement with comparable observations of free and conju-gated forms (Fig. 4). Also, the comparative levels of totalconjugated PAs were higher than free and bound forms.Put was the most abundant PA followed by Spd andSpm, consistent with their normal biosynthesis order.Significant upregulation of nicotine synthesis in L-Orn-

treated tobacco cells was observed; however, there was no

Fig. 2. Free polyamine contents of tobacco cells before and after treatmentSpm, spermine.

change in its production when D-Orn was supplied. The levelof nicotine in L-Orn treatments (10mM) increased fivefoldscompared with control conditions whereas remained constantat all concentrations of D-Orn treatments (Fig. 5).In order to assess whether the observed metabolic effects

of Orn enantiomers is associated with transcript level ofthe responsible genes, we performed the expression studyof SAMDC (Fig. 6). Since the level of Put produced in bothD- and L-Orn-fed cells was almost identical (up to 5mM), itis likely that monitoring the expression of further keyenzymes, for example, SAMDC may shed more light onthe way through which Orn enantiomers function in PAsbiosynthesis. Interestingly, expression of SAMDC was signif-icantly enhanced by D-Orn, whereas L-Orn showed no promo-tional effect on SAMDC expression (Fig. 6). This trend wasquite coincident with the observations about free Spd andSpm where L-Orn showed no positive effect on Spd/Spmlevels (Fig. 2).

DISCUSSIONThese results have shown that not only does L-Orn but also

D-Orn can contribute in the enhancement of the certainpathways for the biosynthesis of metabolites. It is also signif-icant that D-Orn showed no effect on the nicotine level alongwith upregulation of biosynthesis of Spd and Spm. PAs espe-cially Spd and Spm play a vital role in plant responses againstenvironmental stresses like chilling, salinity, and drought.19

Under stress conditions, Spd and Spm keep cell membraneintegrity as compounds with multiple positive charges. ThesePAs are highly positive compounds, and their charges may

with L- and D-ornithine (L- and D-Orn). Put, putrescine; Spd, spermidine;

Chirality DOI 10.1002/chir

Fig. 3. Contents of conjugated polyamines in tobacco cells before and after treatment with L- and D-ornithine (L- and D-Orn). Put, putrescine; Spd, spermidine;Spm, spermine.

Fig. 4. Contents of bound polyamines in tobacco cells before and after treatment with L- and D-ornithine (L- and D-Orn). Put, putrescine; Spd, spermidine.

GHOLAMI ET AL.

counteract with the cationic positive charges in salinity, forexample, with Na+ and Ca2+.20 Changes in the content ofSpd and Spm are shown to be critical for plants cells tosurvive salinity stress condition.21 Therefore, in this investiga-tion, D-Orn has shown to be somehow beneficial for theproduction of necessary metabolites, although D-AAs aremostly considered as futile or toxic for plant cells. Althoughthere have been minimal reports investigating changes inthe metabolism of plant cells with L- and D-AAs, Gamborghad previously used a racemic mixture of Orn and found it

Chirality DOI 10.1002/chir

provided a nitrogen source for plant cell growth.22 The resultspresented here have demonstrated that L-Orn tends toenhance Put and nicotine biosynthesis in tobacco. Given thatL-AA is the natural substrate, L-Orn showed its expected effecton Put and nicotine biosynthesis in the cells. As the productof Orn decarboxylation, Put can participate in two parallelpathways, including Spd, Spm biosynthesis mediated bySAMDC, and nicotine biosynthesis mediated by putrescine-methyl transferase. Upregulated levels of Spd and Spm inD-Orn-treated cells suggest that the SAMDC-dependent step

Fig. 5. Changes in nicotine content of tobacco cells with L- and D-ornithine(L- and D-Orn).

Fig. 6. Transcript level expression of S-adenosylmethionine decarboxylaseof tobacco cells in treatments with L- and D-ornithine (L- and D-Orn).

EFFECT OF ORNITHINE ENANTIOMERS ON THE METABOLISM OF TOBACCO

might have a key role, and Orn enantiomers have differentsuppression or enhancement effects on the rival pathways.According to the gene expression study, transcription levelof SAMDC was highly upregulated by D-Orn, compared withL-Orn. This is in well agreement with levels free of Spd andSpm metabolites. This implies that transcript level enhance-ment of SAMDC has much to do with the observed functionof D-Orn in PAs biosynthesis, and D-Orn’s sensory functionplays a main role in tobacco cells. However, such a sensoryfunction of D-Orn can be relied on several mechanisms, forexample, stereospecific expression of genes transcript or

activities of related enzymes that require further biochemicalstudies. Understanding these trends can be complicated bytranslational or post-translational modification of the resultantproteins, production of conjugated and bound forms of PAs,turnover of PAs via oxidation, and back-conversion reactions.Although illustration of the role of D-Orn in plant metabolismrequires further experiments applying stable isotope labelingallowing us to determine D-Orn metabolism/uptake and PAscatabolic pathway, it can be speculated that the producedSpd and Spm are either direct products of administeredD-Orn or a result of its sensory function on the pathways.Although any sensory function of D-Orn is yet to be reported,some earlier reports have indicated that animal ODC is poten-tially able to metabolize D-Orn.23,24 Therefore, because oflack of any common D-AA metabolizing enzyme in plants,for example, racemases, D-AA oxidases, and D-AA amino-transferases, ODC might be the only known enzyme thatpotentially metabolizes D-Orn in plant cells. Keeping intoaccount that D-Orn is able to specifically enhance Spd andSpm in the plant cell that can be potentially applied forhelping plants to manage PAs content, further studies intothe possible sensory function of D-Orn on the transcript levelof involved genes and metabolism D-Orn/catabolism of PAsby the involved enzymes are warranted.

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