Spatiotemporal oscillations of morphinan alkaloids in opium poppy

15 Pages·2017·1.79 MB·English

by  Mahdi Rezaei

JBiosci Vol.43,No.2, June2018,pp.391–405 (cid:2)Indian Academy of Sciences
DOI: 10.1007/s12038-018-9758-1
Spatiotemporal oscillations of morphinan alkaloids
in opium poppy
MAHDI REZAEI, MOHAMMAD REZA NAGHAVI*, ABDOLHADI HOSSEINZADEH, ALIREZA ABASI
and JABER NASIRI
Division of Plant Molecular Genetics, Department of Agronomy and Plant Breeding,
Agricultural & Natural Resources College, University of Tehran, Karaj, Iran
*Corresponding author (Email, [email protected])
MS received 4 April 2017; accepted 11 April 2018; published online 30 April 2018
Here, a comprehensive endeavor is made to simultaneously scrutinize spatiotemporal oscillations of three imperative
morphinan alkaloids (i.e. thebaine, codeine, and morphine) alongside dynamic transcriptional patterns of TYDC, SalAT,
COR,T6ODM,andCODMgenesindifferenttissuesofPapaversomniferum(i.e.root,bottompartofstem,upperpartof
stem,leaf,capsulewall,andcapsulecontent)overfivedistinguishedontogenicstages(i.e.rosette,budinitiation,pendulous
bud,flowering,andlancing).Apartfrombottomstemandleaf,themaximumthebainecontentoccurredinlancingstage,
while its minimum content did not follow a systematic rhythm, either among six tissues or five various sampling times.
Regardingcodeine,exceptingupperstem,thehighestratiosofcodeinewereobservedatfloweringandlacingstages,while
negligible amounts were overall detected at early stages of plant growth like rosette. Considering morphine, apart from
upperstem,itappearsthatlateontogeneictimesincludinglancingandfloweringarethemostappropriatephasestoachieve
highamountsofmorphine,whileatearlystagestheaforesaidalkaloidpossessedloweraccumulation.Furthermore,allthe
five genes under study, overall, exhibited a variety of transcript levels either among six tissues or five various sampling
times.Interestingly,aconnectionoccurredbetweentranscriptratioofSalATandthebainecontent,suggestingthatthebaine
biosynthesis is coordinated tightly by the enzymatic function of SalATenzyme. Meanwhile, despite low magnitudes of
T6ODM and CODM transcripts in the root-harvested samples at pendulous bud and flowering stages, both codeine and
morphine were surprisingly in acceptable quantities, plausibly owing to the translocation of both alkaloids from the
producing (source) tissues to the roots (sink), known as a phenomenon of ‘source-to-sink transportation’. The results,
altogether, could provide us enough information in acquiring new insights towards potential impacts of spatiotemporal
oscillationsonthemagnitudesofalltheabove-mentionedalkaloidsalongsidetranscriptionratiosofthekeygenesinopium
poppy.
Keywords. BIAsbiosynthesis; geneexpression;morphinan alkaloids;Papaversomniferum;spatiotemporal oscillations
1. Introduction cooperativemanner,andalsoadjustoneanotherthroughtoo
complicatednetworksofcopiousregulatoryand/orstructural
In plant communities, the fact has been well-adopted that genes as well as other known and unknown cellular modu-
both quantity and quality of plant secondary metabolites latory mechanisms. Therefore, it is believed that accurate
(PMSs) may spatially and/or temporally undergone a wide regulation of the genes involved in various biosynthetic
range of variations, worldwide. Such inevitable immediate pathways play a critical role in spatial and temporal-based
disparities in PSMs could be in part attributed the fact of variationsofPSMs(Martinez2002;ZhangandWang2005;
‘sessile nature’ of plants, which in turn enforces them to Yangetal.2012;Ranjbaretal.2015;HashemiandNaghavi
elaborate sensory-response systems versus broad spectrum 2016; Nasiri et al. 2016).
of environmentally favourable/unfavourable alterations By now, the crucial roles of various Papaver species
(Nasiri et al. 2016). In this regards, like other organisms, especially opium poppy (Papaver somniferum L.) as a
plants need to simultaneously recruit diverse physiological, commercialandrenewablerepositoryofBenzylisoquinoline
biochemical and genetical mechanisms in a precise and alkaloids(BIAs)suchasmedicallyimportantpharmaceutical
Electronicsupplementarymaterial: Theonlineversionofthisarticle(https://doi.org/10.1007/s12038-018-9758-1)containssupplementary
material, whichisavailable to authorized users.
http://www.ias.ac.in/jbiosci 391
392 Mahdi Rezaei etal.
drugsofthebaine,papaverine,morphine,codeine,noscapine Eventhough,anumberofnucleotidesequencesencoding
and sanguinarine have been well documented (Dumontet enzymes involved in biosynthetic pathway of BIAs have
and Jordan 2010; Hagel and Facchini 2010; Farrow et al. been so far isolated and characterized, their possible spatial
2012; Hagel and Facchini 2013; Beaudoin and Facchini and temporal variations followed by corresponding
2014), as nearly all of which are utilized widely for the metabolites need to be unraveled yet in details. Here, as a
treatment of moderate to severe and chronic pain. Despite result, a comprehensive endeavor was made to elucidate
such or similar remarkable advantages, the opium poppy is spatiotemporal oscillations of morphinan alkaloids (i.e.,
also regarded as an illegal source of morphine production, thebaine, codeine, and morphine) followed by dynamic
which is simply transformed to heroin (O, O-diacetylmor- transcriptional profiles of one early key gene [i.e. Tyr-
phine) (Beaudoin and Facchini 2014). osine/dopa decarboxylase (TYDC)] followed by four late
ThebiosynthesisandaccumulationofBIAsinP.somniferum crucial genes [i.e. Salotardinol acetyltransferase (SalAT),
havebeenwidelystudiedanddemonstratedcompanioncells, Codeinone reductase (COR), Thebaine 6-O-demethylase
sieve elements and laticifers as three type cells which are (T6ODM), and Codeine O-demethylase (CODM)] in differ-
involvedintheprocess(FacchiniandDeLuca1995;Birdetal. ent tissues of the plant (i.e. root, bottom part of stem, upper
2003;Samananietal.2006).Becauseofcytotoxicitythereof, part of stem, leaf, capsule wall, and capsule content) over
alkaloids broadly accumulate in laticifers in opium poppy five distinguished ontogenic stages (i.e. rosette, bud initia-
(ZieglerandFacchini2008).Theanastomosisoflaticifersinthe tion, pendulous bud, flowering, and lancing).
aerial parts of the plant leads to formation of an articulated
network(Weid etal. 2004).Moreover, the alkaloidcomposi-
tionsmayvaryconsiderably,dependingonthegenotypeunder 2. Materials and methods
study,tissueunderstudy,variousenvironmentaland/orclima-
tologicalcues,differentdevelopmental/phonologicphases,etc. 2.1 Plant materials
(Ranjbaretal.2015;HashemiandNaghavi2016;Rezaeietal.
2016).Therefore,determinationofthealkaloidprofileofeach A local chemotype of P. somniferum was selected and
individualplantwhichwasusedforexpressionanalysisseems initially corroborated cytogenetically to ensure that the
to be an indispensable task (Ziegleretal. 2005). In fact, it is species is P. somniferum (Rezaei et al. 2014). Subsequent
believedthatadditionalinvestigationsdoneedtobeundertaken to verifying its chromosome number (2n = 2x = 22), the
to unravel more mysteries towards biosynthetic pathway of seeds were sterilized first using sodium hypochlorite
morphinanalkaloidseitherattranscriptionallevelsormetabolic (0.5%), washed immediately using distilled water thrice,
biochemistrylevels. and then three of which were planted in a pot containing
Known as the precursor of all other BIAs, (S)-norcoclau- loamandsand(4 kgsoils,pH = 7,EC = 0.78ds/m)inan
rineisnormallyproducedbycondensationoftwo L-tyrosine isolated greenhouse located at the research greenhouse of
derivatives,asthe start point ofBIAs biosynthesis, including Faculty of Agriculture, University of Tehran, Karaj, Iran,
4-hydroxyphenylacetaldehyde (4HPAA) and dopamine (35(cid:3)490 5700 N,50(cid:3)590 2900 E).Tissuesamplingwastaken
through decarboxylation, meta-hydroxylation and transami- place during five distinguished developmental/ontogenic
nation (figure 1). In the next step, (S)-norcoclaurine is con- stages including rosette (figure 2a), bud initiation (fig-
verted into (S)-coclaurine (Morishige et al. 2000; Ounaroon ure 2b), pendulous bud (figure 2c), flowering (figure 2d)
etal.2003)andsubsequentlyistransformedintoacompound and lancing (figure 2e), immediately frozen in liquid
ofN-Methylcoclaurine(Choietal.2001).Afterwards,thanks nitrogen and stored in -80(cid:3)C for further analyses. Nota-
to the hydroxylation of N-Methylcoclaurine, production of bly, depending upon developmental stages, plant samples
3-hydroxy-N-methylcoclaurine is occurred (Pauli and were prepared from root (2 cm from top and 2 cm from
Kutchan1998;Fricketal.2007)andsubsequentlyconverted down parts), 2 cm from lowest part of stem, two terminal
into (S)-reticuline (Morishige et al. 2000). Referring to leaves, 2 cm from topmost part of stem, capsule wall and
biosynthetic pathway (figure 1), thebaine is assumed to be capsule content.
converted intocodeine using a numberofintermediatesteps,
andthroughanenzymatic activityofCODM,this metabolite
is converted into the final product of morphine. Notably, 2.2 RNA isolation and cDNA synthesis
through the second branch, which is assumed to be ‘less
popular’thantheformer(HagelandFacchini2010),thebaine With the purpose of making a fine powder, all the har-
is initially converted into oripavine by CODM activity, and vestedtissuesweregroundusingcoldmortarandpestlein
theresultantconstituentplaysasaprecursortoenzymatically liquid nitrogen. Next, * 100 mg of the resultant powder
generate morphinone. Lastly, the formation of the target was used as starting material for RNA isolation and the
compound of interest, morphine, is eventually ensued, actu- rest stored for alkaloid extraction. RNA isolation, in the
ally via a COR-mediated mechanism. following, was undertaken via a pBIOZOL reagent
Spatiotemporal oscillations of morphinanalkaloids 393
Figure1. Theschematicpathwaysofmajorbenzylisoquinolinealkaloids(BIAs)biosynthesis.Darkgreen(L-Tyrosine):Thestartpoint;
Green: (S)-Reticuline; Blue: Papaverine; Orange: Noscapine; Violet: Sanguinarine; Yellow: Morphine; Pink: Berberine and (S)-
Teterahydropalmitine. The pathways have been deduced from Beaudoin and Facchini (2014). TYDC: tyrosine/DOPA decarboxylase,
3OHase: tyrosine/tyramine 3-hydroxylase, 4HPPDC: 4-hydroxyphenylpuruvate decarboxylase, NCS: norcoclaurine synthase, 6OMT:
norcoclaurine 6-O-methyltransferase, CNMT: coclaurine N-methyltransferase, NMCH: N-methylcoclaurine 30-hydroxylase, 40OMT2: 30-
hydroxyl-N-methylcoclaurine 40-O-methyltransferase (isoform 2), BBE: berberine bridge enzyme, SOMT1: scoulerine 9-O-methyltrans-
ferase,CAS:canadinesynthase,TNMT:tetrahydroprotoberberineN-methyltransferase,CYP82Y1:N-methylcanadine1-hydroxylase,NOS:
noscapine synthase, STOX: (S) tetrahydroxyprotoberberineoxidase, CoOMT: columbamine O-methyltransferase, CFS: cheilanthifoline
synthase,SPS:stylopinesynthase,MSH:N-methylstylopine14-hydroxylase,P6H:protopine6-hydroxylase,DBOX:dihydrosanguinarine
oxidase, SanR: sanguinarine reductase, SalSyn salutaridine synthase, SalR: salutaridine reductase, SalAT: salutaridinol 7-O-acetyltrans-
ferase, T6ODM: thebaine 6-O-demethylase, COR: codeinone reductase, CODM: codeine O-demethylase, N7OMT: norreticuline 7-O-
methyltransferase, 30OHase:uncharacterized 30-hydroxylase, 30OMT: uncharacterized 30-O-methyltransferase.
394 Mahdi Rezaei etal.
Figure2. DifferentdevelopmentalstagesofP.somniferumincluding(a)rosette,(b)budinitiation,(c)pendulousbud,(d)flowering,and
(e)lancing.Atlancingstage,thecapsuleisinitsphysiologicalripeningphase,andiscompletelyreadyfor‘shallowincisionsoncapsules
using asharp knife’ (Khanet al.2007).
(Vinvitrogen) according to manufacturer’s instructions. DNaseI (Fermentas GmbH, St. Leon-Rot, Germany) was
Thereafter, 1.0 lg of the extracted nucleic acid was trea- allowed to heat for 30 min at 37(cid:3)C, and subsequently the
ted by 1.0 lg DNaseI (Thermo Fisher Scientific, USA) to temperature increased up to 65(cid:3)C for 10 min, actually for
confiscate possibleDNAcontamination. For each sample, enzyme inactivation. Henceforward, according to the
RNA concentration was determined first using a Nano- manufacturer’s recommendations, cDNAwas synthesized
Drop(cid:4)ND-1000spectrophotometer,andthesampleswith by adding 200 U/lL Reverse Transcriptase enzyme (Fer-
a 260:280 ratio between 1.9 and 2.1 were employed for mentase, Lithonia) plus 0.5 lg/lL oligo-dT-Primer to the
quantitative analyses. RNA integrity of the samples was mixture and allowed to heat up to 42(cid:3)C for 60 min and
also corroborated through an agarose gel electrophoresis- 70(cid:3)C for 10 min. Lastly, all the synthesized cDNA sam-
based system. First strand cDNA synthesis was accom- plesweredilutedintoa400 ng/lLworkingconcentration,
plished via 1.0 lg treated RNA, as described by (Goojani and stored either at -20(cid:3)C or -80(cid:3)C, for short-term and
et al. 2013): In a word, the reaction mixture containing long-term application, respectively.
Spatiotemporal oscillations of morphinanalkaloids 395
2.3 Quantitative real-time PCR analysis 2.4 Measurement of alkaloids
Gene expression assay was conducted using quantitative With the intention of quantitatively measure alkaloids of
real-time PCR (qRT-PCR) to quantitatively calculate the interest existed in each tissue during various ontogenesis
expression magnitudes of the candidate genes of TYDC, steps, * 100 mg DW samples of the fine powdered bio-
SAT, COR, T6ODM, and CODM, contributed in alkaloids mass were first subjected into 1.0 mL ethanol (70%) and
biosynthetic pathways. PrimerQuest software (Owczarzy allowed to sonicate at 4(cid:3)C for 30 min in 2 mL microfuge
et al. 2008) was applied to design related primer pairs, as tubes.Theresultantmixtures,inthefollowing,wereallowed
fully described in Table S1. To confirm specificity of each to vortex at maximum speed for about one min, centrifuged
primer pair (i.e. amplifying just one aplicon of interest with subsequently at 13000 g, for 10 min at 4(cid:3)C and lastly the
the expected size), each one was theoretically checked supernatants were transferred into the new glass tubes to
using Primer-BLAST software (https://www.ncbi.nlm.nih. utilize as starting materials for alkaloid measurements. In
gov/tools/primer-blast/), and experimentally validated by this regards, an Ion Mobility Spectrometer (IMS-200, TOF
means of conventional PCR. The conventional PCR was Tech Pars) apparatus, which its ionization source is a con-
employed first using a Bio-Rad thermocycler in a total tinuous corona discharge, was employed to quantitatively
volume of 25 lL containing 1X PCR buffer, 0.4 lM of calculate alkaloids contents of each tissue under study. As a
each primer, 0.2 mM of dNTPs, 200 ng of cDNA and 1 well-known technique, IMS offers low detection limit, fast
unit of Taq DNA polymerase (5U/lL). A temperature response, simplicity, and portability which are the main
profile of 5 min at 94(cid:3)C, followed by 35 cycles of 30 s at advantages of IMS compared to the other methods such as
94(cid:3)C, 30 s at the specific annealing temperature for each Gas chromatography. The main parts of the instrument
primer, 60 s at 72(cid:3)C and a final extension at 72(cid:3)C for include two high voltage power supplies, a pulse generator,
10 min. The amplicons, in the following, were elec- IMS cell, a needle for producing the corona, an analog to
trophoresed on 2% agarose gel and visualized through an digital converter and a computer. Details of this technique
ethidium bromide-based staining system. Next, qRT-PCR has been fully described in the previous investigations of
amplification assay was performed via a QIAGEN’s real- (Tabrizchietal.2000;Khayamianetal.2006;Tabrizchiand
time PCR system (Rotor-Gene Q), in a total reaction vol- Shamlouei 2010; Eiceman et al. 2013).The optimized
ume of 20 lL containing 8 lL EvaGreen Master Mix operational conditions for IMS were achieved from
(containing EvaGreen Dye, Solis BioDyn, Tartu, Estonia), (Khayamian et al. 2006), with some minor alterations, as
1.5 lL of diluted cDNA, 0.25 lM of each primer in con- listedinTableS2.Onthesubjectofalkaloidsquantification,
junction with adding RNase free H O. The qRT-PCR was three alkaloids of interest (i.e. thebaine, codeine, and mor-
2
carried out with a temperature profile of 15 min at 95(cid:3)C, phine) were commercially provided from Sigma Aldrich
45 cycles of 15 s at 96(cid:3)C, 20 s at the specific annealing (supplementary figure 1), and utilized then to generate
temperature for each primer, and 20 s at 72(cid:3)C. Both Actin standard curves. To do this, in term of different concentra-
and ELF1a genes were employed and assayed as the two tions of thebaine, codeine, and morphine, the values of
popular reference genes recommended widely for qRT- 0.995, 0.994, and 0.996 were respectively calculated for the
PCR. According to the resultant melt curves, the former correlationcoefficient(R2)ofthestandardcalibrationcurves
exhibited two peaks, while only one sharp peak was oftheaforesaidalkaloids.Notably,withtheaimofchecking
detected for the latter. As a result, based on the melt curve reproducibility, the experiments were performed thrice, and
results as well as previous investigation of (Hagel and the mean ± SE were utilized for comparison survey.
Facchini 2010), only ELF1a was employed as reference
(housekeeping) gene. It is worth noting that, transcript
levels of the ‘leaf samples harvested at rosette stage’ were 2.5 Statistical analysis
set as the reference value, actually to measure transcrip-
tional fold changes. Furthermore, no reverse transcription The data for thebaine, codeine, and morphine contents of
control (NRC) and no-template controls (NTCs) were different plant tissues over various ontogentic phases were
applied as negative controls, and the Cq values of nearly subjected to statistically analyze via a one-way analysis of
C 40 were observed (Nasiri et al. 2016). As described in variance (ANOVA) in acquiring potential significant differ-
our previous works, for each reaction, PCR efficiency was ences(definedasp B 0.05).Theexperimentwasundertaken
automatically calculated by means of QIAGEN’s real-time in terms of a completely randomized design (CRD) with
PCR machine (Rotor-Gene Q), and the values equal or three replications, and the resulting data were consequently
more than 0.8 (80%) were applied for further analyses analyzed using SAS software package (Khattree and Naik
(Nasiri et al. 2016). The experiments were repeated at least 2000). Notably, to statistically make an accurate judgment
thrice, and statistical analysis of gene expression was car- amongdifferenttissues,meancomparisonsurveyswerealso
ried out using REST software (Pfaffl et al. 2002). performed based on Duncan’s multiple range tests (p
396 Mahdi Rezaei etal.
B 0.05).Withrespecttogeneexpressionsection,asdeclared while transcript levels of the samples obtained from lancing
above,threetechnicalreplicationswereappliedfirst,andthe and flowering stages had small amounts of 1.86 and 1.22
relative expression assay was conducted via RESTsoftware fold change, respectively. Upon amplifying capsule wall
(Pfaffletal.2002).Finally,tocalculateandvisuallyperceive samples, the tissues harvested at flowering phase were the
thepossibleassociationsamongtherelativegeneexpression superior(8.96-fold),andlowermagnitudesweretakenplace
data of all the five genes of interest and quantities of three when bud initiation (6.50-fold), lancing (5.43-fold), and
different alkaloids studied here, the ‘corrplot’ and ‘heat- pendulous bud (2.30-fold) were considered. For the last
map.2’ functions was utilized and plotted via the statistical item,capsulecontent,TYDCexhibiteditsmaximumactivity
computingSoftwareRstudio(www.rstudio.com),asanopen at flowering (1.11 fold), and regarding lancing a down-reg-
source integrated development environment (IDE) for R. In ulated behavior (0.12-fold) was detected.
order to determine the significance of each correlation SalAT: Regarding the second gene of interest, SalAT, a
coefficient, the p-values were employed. The smaller the p- number of interesting findings were recorded in each indi-
values, the more significant the corresponding coefficients vidual tissue (table 1): Briefly, comparing to leaf rosette-
become and vice versa. harvestedsamples asreference value, leaf samples collected
inpendulousbudstageencompassedthemaximumvalueof
transcription activity (1.71-fold), and those of pendulous
3. Results bud, flowering, lancing, and bud initiation harbored lower
values of 1.00-, 0.94- and 0.73-fold change, respectively.
3.1 Geneexpressionprofilesduringdifferentontogenic Transcript occurrences of SalAT gene for the root samples
phases in P. somniferum experienced the following descending order of 7.87-, 4.08-,
2.74-, 1.76- and 0.72-fold change at different ontogenic
In general, as could be seen in Table 1, nearly all the five phases of flowering, pendulous bud, rosette, bud initiation,
genes under study exhibited a number of oscillations, as and lancing, respectively. For the bottom stem collected
depending on the tissue and/or sampling phases, some of samples over five different ontogenic phases, the maximum
which were significantly up-regulated, some of which were frequency of the same gene occurred at flowering stage
considerably down-regulated and in some cases no signifi- (12.61-fold), whereas over the ontogenic phases of lancing
cant decrement/increment were observed. In this sense, and (8.80-fold), pendulous bud (5.59-fold), rosette (3.40-fold),
to make a clear judgment for readers, expression profile of andfinallybudinitiation(2.52-fold),transcriptionsystemof
each gene has been individually described as below. Nota- this gene appears to be less active. For the upper stem sec-
bly, as explained above, to quantitatively make relative tion of the plant, the maximum expression frequency of
expression of each gene at different ontogenic phases, ‘leaf SalAT gene occurred at bud initiation (14.51-fold) and
rosette-harvested samples’ were set as reference value. lancing (13.87-fold), albeit for both sampling stage of pen-
TYDC:Onthesubjectofthefirstgeneofinterest,TYDC, dulous bud (8.12-fold) and flowering (5.02-fold) its
thefollowingobservationsoccurredineachtissue(Table 1): expression were still considerable. With the exception of
considering leaf tissue, maximum transcript levels of this capsule wall samples collected at flowering period (3.37-
gene was acquired for the leaves sampled during bud initi- fold), the relative expression proportion of the gene were
ation (2.09-fold change), while regarding those collected quantifiedaslowas1.58,1.20,and0.65foldchangeforbud
over flowering, pendulous bud, and lancing, lower fold- initiation, lancing, and pendulous bud, respectively. For
change values of 1.53, 1.17, and 0.91 were detected, capsulecontent,expressionprofileofthesamegenewasup-
respectively. Taking root samples into account, the samples regulated at flowering time (2.60-fold), while an inconsid-
collected during rosette stage, had a high fold change erablebehavior(0.34-fold)wastakenplaceatlancingphase.
quantity of 111.50, while transcript abundance of the same T6ODM: The highest expression quantity of T6ODM
gene experienced lower fold change ratios of 37.54, 20.13, gene was recorded as 2.95 fold change for the leaf samples
18.61, and 14.17, so long as the collected root samples of of pendulous bud, and regarding sampling times of lancing,
bud initiation, lancing, pendulous bud and flowering were bud initiation, and flowering the inferior amounts of 1.91-,
respectively considered. Unlike root samples, subsequent to 1.55- and 1.46-fold change were detected, respectively
applying bottom stem-derived samples, the maximum tran- (table 1). However, for root samples, T6ODM down-regu-
scription activity of TYDC occurred for bud initiation lated under all the five different ontogenic phases of flow-
(30.02-fold),andthoseharvestedduringlancing(6.71-fold), ering (0.83 fold), bud initiation (0.55-fold), pendulous bud
pendulousbud(2.59-fold),flowering(1.96fold),androsette (0.37-fold), rosette (0.17-fold), and lancing (0.10-fold).
(1.62-fold) were occupied the next descending orders, Considering bottom stem samples, the expression profile of
respectively. Considering upper stem-obtained samples, thesamegenewererecordedas3.44-,2.89-,2.22-,1.99-and
samplescollectedduringpendulousbud(5.44-fold)andbud 0.79-fold change at five sampling times of rosette, bud ini-
initiation(5.22-fold)possessedthehighestexpressionratios, tiation, flowering, lancing, and pendulous bud, respectively.
Spatiotemporal oscillations of morphinanalkaloids 397
Table1. ThetranscriptionratiosofTYDC,SalAT,T6ODM,CODM,andCORgenescontributedtothealkaloidsbiosyntheticpathwayin
eachplant organof P.somniferum at various developmental stages
Organ Developmental stage TYDC SAT T6ODM CODM COR
Rosette 1.0 1.0 1.0 1.0 1.0
bud initiation 2.094ns 0.731ns 1.555ns 1.982* 1.433ns
Leaf
pendulous bud 1.174ns 1.713* 2.947* 9.16* 2.762*
Flowering 1.527ns 1.001ns 1.458* 6.746* 1.892*
Lancing 0.906ns 0.942ns 1.911* 11.517* 2.351*
Rosette 111.498* 2.738* 0.17* 0.033* 3.592*
bud initiation 37.536* 1.763ns 0.552* 0.066* 10.383*
Root
pendulous bud 18.613* 4.082* 0.367* 0.032* 2.758*
Flowering 14.168* 7.873* 0.833ns 0.067* 2.328*
Lancing 20.128* 0.723ns 0.101* 0.129* 2.849*
Rosette 1.618ns 3.395* 3.444* 1.579* 3.892*
bud initiation 30.023* 2.52* 2.894* 1.529* 4.565*
Bottom stem
pendulous bud 2.585ns 5.588* 0.786ns 0.772ns 1.202ns
Flowering 1.958ns 12.613* 2.22ns 2.081* 5.813*
Lancing 6.71* 8.801* 1.997* 0.552* 5.123*
bud initiation 5.221* 14.512v 0.006* 0.004* 1.456ns
upper stem pendulous bud 5.441* 8.119* 3.795* 3.191* 5.987*
Flowering 1.224ns 5.024* 2.414* 0.758ns 7.351*
Lancing 1.864ns 13.865* 5.343* 2.145* 13.796*
bud initiation 6.495* 1.578ns 0.459ns 1.379* 1.048ns
Capsule wall pendulous bud 2.304* 0.653ns 0.361ns 1.068ns 0.359ns
Flowering 8.959* 3.368* 1.393ns 5.317* 5.366*
Lancing 5.434* 1.2ns 1.235ns 5.495* 2.413*
Capsule content Flowering 1.118ns 2.591* 1.86ns 5.551* 2.207ns
Lancing 0.115ns 0.343* 0.61* 3.616* 2.894*
Note: Ratios were calculated relative to the leaf samples harvested at rosette stage as ‘reference value’. Data denote the mean values of three
replicates.TheRed,Yellow,andWhitecolorsindicateup-regulation(pB0.05),down-regulation(pB0.05),andnosignificantdifferencesrelative
tothereference,respectively.ThedatahighlightedwithRed(asup-regulatedgenes)andYellow(asdown-regulatedgenes)colorsaresignificantly
differencethancontrol(i.e.,leafsamplesharvestedatrosettestage)atpB0.05(indictedwith*),whilethosehighlightedwithWhitecolorarenot
statisticallysignificantthancontrol(p[0.05)andindicatedaccordinglywithns.
With regard to the samples of upper stem, the minimum (0.46-fold)pluspendulousbud(0.36-fold),T6ODMactivity
transcript ratios of the gene occurred in bud initiation step declined. For the last tissue, capsule content, the relative
(0.006 fold), while its expression activity seems to be more expression rate of the gene was recorded as 1.86- and 0.61-
provoked at the sampling times of flowering (2.41-fold), fold change at flowering and lancing stages, respectively.
pendulous bud (3.80-fold), and lancing (5.34-fold). Con- CODM:Forleafsamples,theCODMgenecouldmeetits
sidering capsule wall, for both flowering (1.39-fold) and maximum transcript level at lancing (11.51-fold change)
lancing (1.24-fold) a negligible growth was observed com- along with pendulous bud (9.16) phases, and the values of
paredtothereferencevalue,whileconcerningbudinitiation 6.75 and 1.98 fold change were estimated for the leaf
398 Mahdi Rezaei etal.
samples of flowering and bud initiation steps, respectively time up to 7.82 (mg/g DW) for lancing. Leaf thebaine
(table 1). Like T6ODM, small amounts of CODM transcript content appears also to be affected by ontogenesis changes,
levels were recorded, when the root samples at lancing as its maximum and minimum ratios were measured at
(0.13-fold), flowering (0.07-fold), bud initiation (0.07-fold), flowering (1.82 mg/g DW) and pendulous bud (0.63 mg/g
rosette (0.03-fold), and pendulous bud (0.03-fold) were DW) stages, respectively. Similarly, for bottom stem at five
considered. Taking bottom stem into account, among five differentstages,floweringstagewasnominatedastheleader
differenttimesamplings,twoofwhichweredown-regulated (4.70 mg/gDW),andbudinitiation(1.13 mg/gDW)hadthe
[i.e., pendulous bud (0.77-fold), and lancing (0.55-fold)], minimumvalue.Forupperstem,threesamplingdatesofbud
while the rest possessed more transcript levels [i.e., flower- initiation, pendulous bud and flowering had nearly equal
ing (2.08-fold), rosette (1.58-fold), and bud initiation (1.53- thebainequantities,whilelancingexhibitedmoreamountsof
fold)].Unlikebottomstem,thelowestexpressionactivityof 5.91 mg/g DW. Similarly, thebaine content of capsule wall-
the same gene for the tissues belonging to upper stem harvested samples at lancing time was the superior
occurred at bud initiation (0.004-fold) and flowering (0.76- (6.04 mg/g DW), while for the samples belonging to the
fold),whilehigherproportionswereachievedforpendulous otherontogenicstagessuchasflowering,pendulousbudand
bud(3.19-fold)andlancing(2.15-fold).Consideringcapsule bud initiation lower amounts of 4.60 mg/g DW, 2.20 mg/g
wall samples, transcript incidence of this gene was slightly DW, and 0.81 mg/g DW were measured, respectively.
increased at both bud initiation (1.07-fold) and pendulous Considering capsule content, this ratio was nearly amelio-
bud (1.38-fold), and gradually intensified, reaching to the ratedas2.48 mg/gDWand2.65 mg/gDWatbothflowering
maximum levels of 5.32- and 5.50-fold change at flowering and lancing times, respectively.
and lancing times, respectively. Lastly, for the capsule con- Codeinecontent:Regardlessofsomefluctuations,allthe
tent samples collected at either flowering (5.55-fold) or tissuesoftheplantstudiedherecontainedcodeine,atleastin
lancing (3.62-fold), remarkable transcripts were detected. one or more ontogenic phases, as detailed below (figure 4):
COR: Considering the first tissue, leaf, the highest pro- in root, codeine was only detected at flowering stage
portionofCORgenewasobservedforpendulousbud(2.76- (2.83 mg/gDW)followedbyasmallquantityof0.56(mg/g
fold)andlancing(2.35-fold),whereasatfloweringfollowed DW)forpendulousbud.Forbottomstem,however,codeine
by bud initiation lower transcript levels of 1.89- and 1.43- was measured in all the five sampling times, ranging from
fold change were acquired, respectively. Unlike leaf, sig- 2.37(mg/gDW)atrosetteto4.27(mg/gDW)forflowering.
nificant transcription growth was recorded for the root Similarly, taking leaf tissue into consideration, the codeine
samples harvest at rosette (3.59-fold) and particularly bud quantity ranged from 1.73 (mg/g DW) for bud initiation up
initiation (10.38-fold), whilst for the rest three sampling to 5.34 (mg/g DW) for flowering time. Considering upper
phases of lancing (2.85-fold), flowering (2.33-fold), and stem, exclusive of rosette phase whose codeine content was
pendulous bud (2.76-fold), activity thereof declined, albeit notdetectable,considerablemagnitudesofthesamealkaloid
all the values were still beyond reference value. On the was quantified for the remaining three sampling times,
subject of bottom stem, expression profile of the same gene rangingfrom4.37(mg/gDW)forbudinitiationstepto6.98
up-regulated, as flowering (5.81-fold) and pendulous bud (mg/g DW) for pendulous bud. On the subject of capsule
(1.20-fold) samples had the maximum and minimum tran- wall, again, no detectable codeine was observed at rosette
script levels. Apart from bud initiation (1.46-fold), upon stage, whilst conspicuous values of codeine were detected
applying upper stem, remarkable transcription activity of for the samples collected at bud initiation (2.86 mg/g DW),
CORgeneoccurredforpendulousbud(5.99-fold),flowering pendulous bud (4.15 mg/g DW), flowering (4.84 mg/g
(7.35-fold) and lancing (13.80-fold). Regarding capsule DW), and lancing (5.37 mg/g DW) stages. Regarding cap-
wall, owing to having a value of 5.37 fold change, flower- sule content, acceptable amounts of this alkaloid were
ing-harvestedsamplescouldsurpasstherestthreeontogenic recorded at both flowering (4.37 mg/g DW) and lancing
times of lancing (2.41-fold), bud initiation (1.05-fold) and (2.73 mg/g DW) phases, could be accordingly regarded as
pendulousbud(0.36-fold).Asthecapsulecontentharvested one of the valuable source in generating codeine.
atfloweringandlancingweretakenintoaccount,thevalues Morphine content: Similar to the codeine, there was no
of 2.21- and 2.89-fold change were achieved, respectively. detectable morphine when the root samples at rosette, bud
initiation, and lancing stages were taken into account, albeit
a tiny amount of 0.38 mg/g DW followed by a significant
3.2 Effect of ontogenic changes on the accumulation rate of 4.23 mg/g DW were quantified for pendulous bud
of thebaine, codeine, and morphine and flowering phases, respectively (figure 5). Regarding
leaf, the maximum and minimum quantities of morphine
Thebaine content: As figure 3 is shown, all the root sam- were detected atlancing(5.43 mg/g DW) andbud initiation
ples harvested at five different ontogentic phases accumu- (1.35 mg/g DW), while concerning bottom stem, both
lated thebaine, varied from 1.96 (mg/g DW) at pendulous lancingtogetherwithflowering(4.08 mg/gDW)androsette
Spatiotemporal oscillations of morphinanalkaloids 399
Thebaine
9
a
8
)W
D 7
rg b a a
/g 6
m
( c a b
tn 5
tne 4 d b b b b
eiabocenh 23 e cddbc abcb c aabc d c a a
T 1
0
Root Bo(cid:2)om stem Leaf Capsule wall Upper stem Capsule content
Rose(cid:2)e Bud ini(cid:3)a(cid:3)on Pendolous bud Flowering Lancing
Figure3. Thethebainecontentsofroot,bottompartofstem,leaf,capsulewall,upperpartofstemandcapsulecontentinP.somniferum
over five different ontogenic phases of rosette, bud initiation, pendulous bud, flowering, and lancing. Data indicate the mean values of
quantities of three replication. Different letters (a, b, c, d, and e) indicated above the bar represent statistically significant difference at
p B0.05 (Duncan’smultiple range test).
8 Codeine
a
)W 7
D
rg 6 a a
/g a b b
m 5 a b a b a
(
tne 4 abbc bc
tn a c b b
o 3 c
ce d d
n 2
ie
d b
o 1
C
0
Root Bo(cid:2)om stem Leaf Capsule wall Upper stem Capsule
content
Rose(cid:2)e Bud ini(cid:3)a(cid:3)on Pendolous bud Flowering Lancing
Figure4. Thecodeinecontentsofroot,bottompartofstem,leaf,capsulewall,upperpartofstemandcapsulecontentinP.somniferum
over five different ontogenic phases of rosette, bud initiation, pendulous bud, flowering, and lancing. Data indicate the mean values of
quantities of three replication. Different letters (a, b, c, and d) indicated above the bar represent statistically significant difference at
p B0.05 (Duncan’smultiple range test).
(2.54 mg/g DW) phases possessed the highest and lowest 3.3 Correlationbetweenalkaloidsandgeneexpression
ratio of morphine, respectively. The metabolic data of the data
upperstemrevealedthatthemaximumquantityofmorphine
couldoccuratpendulousbud(11.11 mg/gDW),albeitother So as to statistically identify possible correlations between
three ontogenic phases of flowering (8.15 mg/g DW), bud alkaloids accumulation and relative expression data of all the
initiation (7.49 mg/g DW) and lancing (6.28 mg/g DW) five genes of interest in different tissues over different ontho-
seems to be still ‘acceptable alternative options’. Consider- genicphases,correlationanalysiswascarriedout.Toendthis,
ing capsule wall, sampling at flowering gave the maximum for each tissue a heatmap plot was generated via Rstudio
quantity of morphine (8.98 mg/g DW), while collection at programandtheresultantgraphswereappliedthentovisually
bud initiation (3.88 mg/g DW) provided lowest amounts. observe the potential associations between alkaloids accumu-
Taking capsule content into account, morphine contents at lations and transcripts data. Notably, to compute correlation
both flowering and lancing stages were substantially mea- coefficient(q),foreachindividualtissue,themeanvaluesofall
sured as 7.10 mg/g DWand 4.80 mg/g DW, respectively. the ontogenic phases were together selected.
400 Mahdi Rezaei etal.
12 Morphine a
)W
D
rg/ 10 a b
g bc
m 8 a
( b c
ntteno 6 a a a a a cbc b
c ab
enihrop 24 b c b bcc b
M
0
Root Bo(cid:2)om stem Leaf Capsule wall Upper stem Capsule
content
Rose(cid:2)e Bud ini(cid:3)a(cid:3)on Pendolous bud Flowering Lancing
Figure5. Themorphinecontentsofroot,bottompartofstem,leaf,capsulewall,upperpartofstemandcapsulecontentinP.somniferum
over five different ontogenic phases of rosette, bud initiation, pendulous bud, flowering, and lancing. Data indicate the mean values of
quantitiesofthreereplication.Differentletters(a,b,andc)indicatedabovethebarrepresentstatisticallysignificantdifferenceatp B0.05
(Duncan’s multiple rangetest).
As figure 6aisshown,regardingleaf sampleharvestedat occurred(Levyetal.1988).Furthermore,atthesamestudy,
five different sampling times, no significant associations different parts of the root possessed considerable variations
were statistically acquired between the genes under study for thebaine accumulation, suggesting that thebaine pro-
and three alkaloids of choice, albeit a number of high cor- duction/accumulation could be either tissue- or ontogenic-
relations were achieved. However, taking root samples har- dependentphenomenon(Levyetal.1988).Similarly,inour
vested at five different sampling times into account recent study working on various organs of P. bracteatum, it
(figure 6b), between SalAT transcripts and morphine plus was overallrecoveredthat thebaine accumulationinroots is
codeine rates significant correlations were achieved more frequent at flowering (1.99 mg/g FW) and lancing
(q = 0.9, P\0.05; q = 0.95, P\0.05). Furthermore, stages(2.02 mg/gFW),whileinitialdevelopmentalstagesof
betweenCODMtranscriptsandthebaineaccumulationinthe the plant like annual (0.97 mg/g FW) and perennial rosette
aforesaid tissue significant correlation was achieved (1.10 mg/gFW)phasesfailedtoexhibitremarkableamounts
(q = 0.93, P\0.05). On the subject of bottom stem sam- of this metabolite (Rezaei et al. 2016). Notably, except for
ples harvested at five different sampling times (figure 6c), a leaf tissue, the remaining parts of the plant had no
strong correlation was only observed between SalAT tran- detectable amounts of the metabolite (Rezaei et al. 2016).
scriptsandtebaineratio(q = 0.98,P\0.001).Considering Here,resemblingourpreviouswork(Rezaeietal.2016),the
upper stem samples harvested at four different sampling highest thebaine accumulation occurred for the roots
times (figure 6d), statistically no significant correlations belonging to flowering and lancing stages, albeit quantities
wereachieved.Regardingcapsulewallsamplesharvestedat thereof were approximately three and four times more than
four different sampling times, despite the fact that high ourearlier record.Suchobservations connotedthatthebaine
values of correlation ratios were obtained, no ones were production could be also a species-dependent phenomenon.
statistically significant (figure 6e). At the study of (Gu¨mu¨s¸c¸u¨ et al. 2008), thebaine content of
capsule-derived samples of 99 lines of P. somniferum har-
vested in mid-July ranged from 0.005% to 0.134%, with a
4. Discussion mean value of 0.041%. In addition, considering alkaloids
content within total alkaloids, the proportion of thebaine
As noted above, thebaine accumulation varied not only varied from 1.9% to 18.1%, with a mean value of 5.66%
among different tissues sampled from ‘the same ontogenic (Gu¨mu¨s¸c¸u¨ et al. 2008). In a number of poppy cultivars
time’, but also over various ontogenic stages of ‘the same originated from England, Hungary, Afghanistan, India, and
tissue’, which could somewhat comparable to the earlier Thailand, thebaine concentration ranged as 0.00–0.01%,
works. For instance, according to (Levy et al. 1988), the 0.00–0.05%, 0.00–0.01%, 0.00–0.03%, and 0.00–0.01%,
ratio of thebaine in the roots of P. bracteatum undergonean respectively (Berna´th et al. 1988). Meanwhile, thebaine
increasing behavior from vegetative rosette to flowering content of the latex of 1470 individual plant samples
phase, reaching to a peak at flower bud (Levy et al. 1988). belonging to 98 germplasm lines was recorded as 0.52% to
Nonetheless, over reproductive phase in conjunction with a 7.95%, with a mean value of 2.27% ± 0.116 (Shukla et al.
trifling increment at the end of flowering a decrement 2006). Using 122 accessions of P. somniferum, Yadav et al.