Research Article Nitrate Promotes Capsaicin Accumulation in … · 2018. 10. 25. · Research Article Nitrate Promotes Capsaicin Accumulation in Capsicum chinense Immobilized Placentas

Research ArticleNitrate Promotes Capsaicin Accumulation inCapsicum chinense Immobilized Placentas

Jeanny G Aldana-Iuit12 Enrique Sauri-Duch1 Mariacutea de Lourdes Miranda-Ham2

Lizbeth A Castro-Concha2 Luis F Cuevas-Glory1 and Felipe A Vaacutezquez-Flota2

1 Instituto Tecnologico de Merida Km 5 Carretera Merida-Progreso 97118 Merida YUC Mexico2Unidad de Bioquımica y Biologıa Molecular de Plantas Centro de Investigacion Cientıfica de Yucatan Calle 43 No 130Chuburna de Hidalgo 97205 Merida YUC Mexico

Correspondence should be addressed to Felipe A Vazquez-Flota felipecicymx

Received 12 November 2014 Revised 1 December 2014 Accepted 4 December 2014

Academic Editor Akio Hiura

Copyright copy 2015 Jeanny G Aldana-Iuit et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

In chili pepperrsquos pods placental tissue is responsible for the synthesis of capsaicinoids (CAPs) the compounds behind theirtypical hot flavor or pungency which are synthesized from phenylalanine and branched amino acids Placental tissue sectionsfrom Habanero peppers (Capsicum chinense Jacq) were immobilized in a calcium alginate matrix and cultured in vitro eithercontinuously for 28 days or during two 14-day subculture periods Immobilized placental tissue remained viable and metabolicallyactive for up to 21 days indicating its ability to interact with media components CAPs contents abruptly decreased during thefirst 7 days in culture probably due to structural damage to the placenta as revealed by scanning electron microcopy CAPs levelsremained low throughout the entire culture period even though a slight recovery was noted in subcultured placentas Howeverdoubling the mediumrsquos nitrate content (from 40 to 80mM) resulted in an important increment reaching values similar to thoseof intact podrsquos placentas These data suggest that isolated pepper placentas cultured in vitro remain metabolically active and arecapable of metabolizing inorganic nitrogen sources first into amino acids and then channeling them to CAP synthesis

1 Introduction

In plants the placenta is the filmy tissue that holds the seedsinside the fruit In the Capsicum genus it also produces andaccumulates capsaicinoids (CAPs) compounds responsiblefor the burning flavor of peppers [1] CAPs are formedthrough the convergence of two biosynthetic pathways onefrom phenylalanine and the other from branched aminoacids either valine or leucine [2ndash5] Due to the interest inCAPs biosynthesis in vitro cell cultures from different Cap-sicum species have been employed as a model to study thebasis for its regulation however nondifferentiated cell sus-pensions accumulate very low CAP amounts compared tointact placental tissues [6 7] which ismaybe due to the lack ofthe required specialized structures In intact placentas CAPsare stored in vesicles derived from the epithelial cells [1]Since CAP biosynthesis is exclusively located in the placentaltissue the use of sections of this tissue cultured in vitro has

been envisioned as an alternative system [7 8] Two methodsfor the in vitro culture of pepper placentas have been usedfree and immobilized tissues Free placentas cannot maintaintheir integrity as they become swollen and disintegrate aftershort periods in culture This effect can be avoided byimmobilizing the tissue [9] Tissue immobilization consistsin entrapping sections within an inert support keeping italive and functional in a medium with mineral salts andorganic compounds under sterile conditions Immobilizedtissues will remainmetabolically active although growth anddevelopment have been arrested This technique has beenwidely employed for the production of plant fine chemicalsas well as for their functional preservation [9] On the otherhand mineral nutrition can affect CAPs accumulation andthence pungency in chili peppersMoreover these effects canbe exerted at various levels such as modifying the biosyn-thetic ability of the participating tissues or promoting theirbiomass accumulation [10 11] Three nitrogenous molecules

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 794084 6 pageshttpdxdoiorg1011552015794084

2 BioMed Research International

O CH=O

HO

HO

OH

OH

HO

O

O

CoAS

O

O NH

O Capsaicin

COOH

COOH

O

O

Phenylalanine

Cinnamic acid

PAL Valine

BCAT

AMTHO

O

GABA

CS

NH2

NH2

NH2

120572-Ketoisovalerate

H3C

H3C

H3C

8-Methyl-6-nonenoyl-CoA

CH2ndashNH2

Vanillin

vanillylamine

Figure 1 Involvement of nitrogen or nitrogenous metabolites in capsaicin biosynthesis AMT vanillin aminotransferase BCAT branchedamino acid aminotransferase PAL phenylalanine ammonia-lyase

are involved in CAPs synthesis (Figure 1) These are Pheny-lalanine which provides the phenolic moiety a branchedamino acid either valine or leucine that contributes with thelateral aliphatic chain and 120574-aminobutyric acid (GABA) theamino donor required to form vanillylamine from vanillin([12] Figure 1) Hence as expected CAP contents in pepperpods respond to external nitrogen availability In fact a posi-tive correlation between nitrate and CAPs contents occurs inplacentas from Habanero pepper pods [11] To further assessthe dependence of CAP biosynthesis on nitrogen availabilityisolated Habanero pepper placentas were cultured in vitroand exposed to different nitrate doses Previous to nitrogentreatments placentas were immobilized in calcium alginatein order to keep tissue integrity and metabolic functionalitywas evaluated

2 Materials and Methods

21 Biological Materials Placentas were obtained from podsof an orange accession of Habanero pepper (Capsicumchinense Jacq) collected from commercial plantations in

Yucatan Mexico [13 14] Cell suspensions ofC chinenseweremaintained at the same conditions described previously [15]

22 Tissue Immobilization Process Immature pods (16ndash20days after anthesis ca 3times2 cm longtimeswide)were used shortlyafter harvest Pods were disinfested with ethanol (80) andcommercial bleach (50 equivalent to 3mgL of sodiumhypochlorite) before placentas were excised with scalpeland tweezers Placentas were kept in sterile water prior toimmobilization or transfer to the culturemedium Sections ofplacental tissue (025 cm2) were suspended in a beaker con-taining 25 sodium alginate (Sigma) at room temperatureinside a flow cabinet with constant and gentle stirring in orderto distribute them homogenously in the vessel To obtain thealginate beads a blunt-tip 10mL glass pipette was used todrop ca 1mL of this suspension into cold 1 CaCl

2under

constant gentle stirring Immobilized placentas were allowedto stand for 90 minutes at 4∘C washed with cold distilledwater to eliminate excess calcium and transferred to 250mLErlenmeyer flasks containing 40mL of Murashige and Skoog(MS) mediumwithout growth regulators Each flask received

BioMed Research International 3

(a) (b) (c)

Figure 2 Scanning electron micrographs of immobilized placentas after 7 (a) 14 (b) and 21 (c) days in in vitro culture Circle-enclosed areaswith the presence of blisters either intact (a) or damaged (b and c)

ca 40 alginate beads with tissue sections entrapped and werekept for a 28-day culture period either continuously or witha renewal of medium after the first 14 days

23 Nitrate Treatments Nitrate in MS medium is suppliedas a mixture of NH

4NO3and KNO

3 accounting for a

total concentration of 40mM In order to promote CAPsaccumulation immobilized placentas were exposed to dif-ferent nitrate doses 20 40 (normal content) 60 or 80mMPotassium nitrate was used in the 20 and 40mM treatmentsso no potassium compensation was required in these treat-ments Nevertheless ammonium content was compensatedas NH

4Cl when total nitrate was reduced to 20mM To

avoid an excess of potassium ions extra nitrate in 60 and80mM treatments was supplied as NaNO

3 Any side effects

resulting from extra chloride and sodium ions were analyzedby exposing the immobilized placentas to 20mMNaCl addedto standardMSmedium After 14 days immobilized placentasections were either subcultured to fresh media with thesame nitrate doses or kept in the original media for an extra14 days In both cases total culture period was 28 daysSimilar experiments were performed with cell suspensionsfor comparative analysis Two-week old cell cultures weretransferred to fresh MS medium with 20 40 (control) 60 or80mMnitrate adjusting to one gram of cells (FW) per 40mLof media contained in 250mL Erlenmeyer flasks

24 Analytical Procedures After treatments immobilizedplacentaswere collected frozen in liquid nitrogen and kept atminus80∘C until analysis To estimate tissue viability throughoutthe culture period total soluble sugar and amino acids werequantified in aqueous extracts following standard procedures[8] Phenylalanine ammonia-lyase (PAL) activity that cat-alyzes the first reaction leading to CAP biosynthesis wasanalyzed [15] to evaluate possible effects on this pathwayProtein extracts were obtained as follows beads were groundto a fine powder and mixed with appropriate extraction

buffers [8 15]The slurry was first filtered through four layersof cheesecloth and then centrifuged to eliminate alginateresidues CAPs were evaluated in methanolic extracts afterchromatographic separation as described previously [16]

25 Scanning Electron Microscopy (SEM) Immobilized pla-cental tissue was excised from pods with scalpel and tweezersand fixed with FAA (10 formaldehyde 50 ethanol and 5acetic acid) as described elsewhere [17] Afterwards tissueswere dehydrated with an increasing ethanol series (from30 to 100) and solvent-exchanged with liquid CO

2 and

dried to critical-point (Penten Vacuum Desk) Dehydratedplacental tissues were then mounted on aluminum stubs andtwice coatedwith gold emulsion (Sputter-coater Sandri-795)Section surface was examined using a JEOL 6360LV scanningelectron microscope (SEM)

26 Statistical Analysis Data were analyzed using one- ortwo-way ANOVA and means were compared using TukeyrsquosHSD test at 119875 lt 005 with the Statsoft Statistica 7 packageResults shown correspond to triplicates taken fromone singleexperiment since trends were similar in two independentexperiments Mean values are presented with their standarddeviation

3 Results and Discussion

Immobilized placentas were cultured in vitro for either a sin-gle continuous 28-day period (nonsubcultured) or throughtwo 14-day culture periods (one subculture)

In intact placentas CAPs are accumulated in vesi-cles derived from swollen epithelial cells (Figure 2(a) [1])The structure of such vesicles was observed by electronmicroscopy (Figure 2) Although most of these vesicles werepreserved in encapsulated placentas manipulation may havecaused some physical damage (Figure 2(b)) No further dam-age to these structures was recorded throughout the culture


Time (d)0 7 14 21 28

0

250

500

750

1000

1250

Continuous cultureSubculture

Tota

l sug

ars (120583

g gminus1

DW

)

(a)

Time (d)0 7 14 21 28

0

200

400

600


Tota

l am

ino

acid

s (120583

g gminus1

DW

)

(b)

Figure 3 Total sugars (a) and amino acids (b) contents in immobilized placentas cultured in vitro for continuous 28 days (continuous lines)or with medium renewal after 14 days (dashed lines subculture) Means of 5 replicates plusmn SD

period neither the formation of new ones (Figures 2(b) and2(c)) Nevertheless CAPs recovered from the medium werein very low concentrations perhaps due to their oxidationas has been previously proposed [18] This fact may explainthe cause of lower CAPs contents in immobilized placentaswhen compared to the intact tissue excised from pepperpods (ca 05 versus 66mg gminus1DW) In order to determine ifimmobilized placentas were still metabolically active solublesugars and amino acids contents were analyzed in both thosemaintained in a 28-day continuous culture or with a 14-daysubculture (Figure 3) During the first seven days of culturesugars remained unchanged compared to the initial contentsincreasing shortly after However further increase was onlynoticed in subcultured placentas (Figure 3(a)) Soluble aminoacids sharply decreased in the first seven days and thenrecovered their initial values In the case of those placentasthatwere not subcultured the contents declined again sharply(Figure 3(b))

In this way renewal of the medium might have stoppedthe decline in these metabolites levels suggesting that theencapsulated tissues under in vitro conditions were able tointeract with the surrounding medium Moreover nonde-tached placentas from pods stored under similar conditionsto those in in vitro cultures (culture room conditions) did notmaintain the initial values through similar time periods (datanot shown) implying that decay in metabolic activity wasoccurring in those tissues that were not receiving an externalsupply of nutrients Interestingly CAPs accumulation alsoincreased in response to medium renewal (Figure 4) Pheny-lalanine ammonia-lyase (PAL) activity the first enzyme of thephenylpropanoid pathway and thus initiating the formationof the phenolic moiety of CAPs decreased to a fourth ofthat found in intact placentas during the first seven days inculture (Figure 5) However PAL activity levels reverted totheir initial values in intact tissues after 14 days although

Time (d)0 7 14 21 28

0

200

400

600

800

1000

1200

1400


Caps

aici

noid

s (120583

g gminus1

DW

)

Figure 4 Capsaicinoid content in immobilized placentas culturedin vitro for 28 days continuous lines) or with medium renewal after14 days (dashed lines subculture) Means of 5 replicates plusmn SD

such levels were not maintained afterwards (Figure 5) Themodulation pattern shown by PAL suggests that immobilizedplacentas might maintain the ability to produce the phenoliccompounds required for CAPs biosynthesis although nodirect correlation between PAL activity (Figure 5) and CAPsaccumulation (Figure 4) was observed This result contrastswith what was found for suspension cells from C annuumwhere an increase in PAL activity correlated to an importantaccumulation of both CAPs and phenolic intermediaries [7]suggesting that regulatory controls for CAPs synthesis inintact placentas may be exerted at different points than in cellcultures [19]


Time (d)0 7 14 21 28

0

20

40

60

80

100

120

PAL

activ

ity(120583

g ci

nnam

ic ac

idm

inminus1

mg

prot

minus1)

Figure 5 Phenylalanine ammonia-lyase specific activity in intactplacentas and after 7 14 21 and 28 days of in vitro culture Means of5 replicates plusmn SD

Time (d)0 7 14

0

300

600

900

1200

1500

1800

Pepper placenta40mM

60mM80mM

Caps

aici

noid

s (120583

g gminus1

DW

)

Figure 6 Capsaicinoid content in placentas cultured in vitro underthree nitrogen regimes for 7 or 14 days White bars 40mM nitrategray bars 60mM nitrate and black bars 80mM nitrate Means of 5replicates plusmn SD Dashed bar at 0 days represents initial capsaicinoidcontents

31 Nitrate Increases CAPs Accumulation in Immobilized Pla-centas of Habanero Pepper Immobilized pepper placentaswere able to remain metabolically active and to produceCAPs throughout a 21-day culture period (Figures 3 and 4)When nitrogen content in the media was modified culturedplacentas showed concomitant variations in CAPs contentsAfter seven days in culture placentas exposed to half the nor-mal dose of MS nitrate (20mM) accumulated undetectableamounts of CAPs (data not shown) In contrast when thefull MS nitrate content (40mM) was employed placentasaccumulated up to 03mgCAP gminus1DW A sharp increase inCAPs level was detected when available nitrate increasedto 60mM (64mg gminus1DW) or 80mM (156mg gminus1DW) forseven days (Figure 6) Longer exposures to these nitrate

Time (days)

0

20

40

60

80

100

0 7 14 21

80mM

60mM20mM

Caps

aici

n (120583

g gminus1

DW

)

40mM(control)

Figure 7 Capsaicin content in C chinense cell cultures exposed todifferent nitrate doses Two-week cultures were transferred to freshmedium with 20 40 (control) 60 or 80mM nitrate Means of 5replicates plusmn SD

levels were detrimental to CAPs accumulation since therewere decreases of over 80ndash85 in the levels observed beforeThese results confirm that immobilized placentas culturedin vitro were able to synthesize CAPs and suggest thatexternally supplied nitratemay be converted into the requiredamino acids for this process Activation of primary nitrogenassimilation in response to modification of the nitrogensource has been demonstrated in placental tissues culturedin vitro [8] It is noteworthy to point out that the increase inCAPs levels when nitrate concentration was increased from60 to 80mM was lower than that observed between 40 and60mM of nitrate Interestingly undifferentiated C chinensecell cultures did not respond in a similar fashion to variationsin the nitrogen source (Figure 7) This may imply that otherfactors than nitrogen availability such as the presence oforganized tissue could limit CAPs accumulation in in vitroconditions

4 Concluding Remarks

Immobilized placentas of Habanero pepper could be keptviable and metabolically active for up to 21 days under theassayed conditions Interestingly as it has been recordedfor intact placentas in whole pepper pods [11] in vitrocultured placentas increased their CAP contents when nitrateavailability increased (Figure 6) These observations suggestthat isolated placentas can interact with the components ofthemedium and that inorganic nitrogen can be incorporatedfirst into amino acids and then to CAPs In this sensean active primary assimilation of nitrogen into amino acidshas been determined in placentas of Habanero peppers [8]Therefore given the proper conditions placental tissuesare able to autonomously function to maintain complexmetabolic pathways such as those involved in secondarymetabolism


Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Theauthors thankMS A F Ku-Gonzalez (scanning electronmicroscopy) MS M Monforte-Gonzalez (capsaicin analy-sis) and Ing F Contreras-Martın (culture of pepper plants)This study was supported by CONACyT (Mexico) JeannyGAldana-Iuit receivedCONACYTandDGEST scholarshipsfor doctoral studies

References

[1] C Stewart Jr M Mazourek G M Stellari M OrsquoConnell andM Jahn ldquoGenetic control of pungency in C chinense via thePun1 locusrdquo Journal of Experimental Botany vol 58 no 5 pp979ndash991 2007

[2] B C N Prasad H B Gururaj V Kumar P Giridhar and GA Ravishankar ldquoValine pathway is more crucial than phenylpropanoid pathway in regulating capsaicin biosynthesis inCapsicum frutescens Millrdquo Journal of Agricultural and FoodChemistry vol 54 no 18 pp 6660ndash6666 2006

[3] B C N Prasad H B Gururaj V Kumar et al ldquoInfluence of 8-methyl-nonenoic acid on capsaicin biosynthesis in in-vivo andin-vitro cell cultures of Capsicum spprdquo Journal of Agriculturaland Food Chemistry vol 54 no 5 pp 1854ndash1859 2006

[4] H G Nunez-Palenius and N Ochoa-Alejo ldquoEffect of pheny-lalanine and phenylpropanoids on the accumulation of capsai-cinoids and lignin in cell cultures of chili pepper (Capsicumannuum L)rdquo In Vitro Cellular amp Developmental BiologymdashPlantvol 41 no 6 pp 801ndash805 2005

[5] F Vazquez-Flota M D L Miranda-Ham M Monforte-Gonzalez G Gutierrez-Carbajal C Velazquez-Garcıa and YNieto-Pelayo ldquoBiosynthesis of capsaicinoids the pungent prin-ciple of peppersrdquo Revista Fitotecnia Mexicana vol 30 no 4 pp353ndash360 2007

[6] A R Altuzar-Molina J A Munoz-Sanchez F Vazquez-FlotaM Monforte-Gonzalez G Racagni-Di Palma and S M THernandez-Sotomayor ldquoPhospholipidic signaling and vanillinproduction in response to salicylic acid and methyl jasmonateinCapsicum chinense J cellsrdquoPlant Physiology andBiochemistryvol 49 no 2 pp 151ndash158 2011

[7] C Aza-Gonzalez H G Nunez-Palenius and N Ochoa-AlejoldquoMolecular biology of capsaicinoid biosynthesis in chili pepper(Capsicum spp)rdquo Plant Cell Reports vol 30 no 5 pp 695ndash7062011

[8] W D R Ancona-Escalante F M Baas-Espinola L A Castro-Concha F A Vazquez-Flota M Zamudio-Maya and M DL Miranda-Ham ldquoInduction of capsaicinoid accumulation inplacental tissues of Capsicum chinense Jacq requires primaryammonia assimilationrdquo Plant Cell Tissue and Organ Culturevol 113 no 3 pp 565ndash570 2013

[9] T S Johnson G A Ravishankar and L V Venkataraman ldquoInvitro capsaicin production by immobilized cells and placentaltissues of Capsicum annuum L grown in liquid mediumrdquo PlantScience vol 70 no 2 pp 223ndash229 1990

[10] J L Guil-Guerrero C Martınez-Guirado M del Mar Rebol-loso-Fuentes and A Carrique-Perez ldquoNutrient composition

and antioxidant activity of 10 pepper (Capsicum annuun) vari-etiesrdquo European Food Research and Technology vol 224 no 1pp 1ndash9 2006

[11] M Monforte-Gonzalez A Guzman-Antonio F Uuh-Chimand F Vazquez-Flota ldquoCapsaicin accumulation is related tonitrate content in placentas of habanero peppers (Capsicum chi-nense Jacq)rdquo Journal of the Science of Food and Agriculture vol90 no 5 pp 764ndash768 2010

[12] H B GururajM N Padma P Giridhar andG A RavishankarldquoFunctional validation ofCapsicum frutescens aminotransferasegene involved in vanillylamine biosynthesis using Agrobac-terium mediated genetic transformation studies in Nicotianatabacum and Capsicum frutescens calli culturesrdquo Plant Sciencevol 195 pp 96ndash105 2012

[13] L A Castro-Concha I Canche-Chuc and M D L Miranda-Ham ldquoDetermination of antioxidants in fruit tissues from threeaccessions of habanero pepper (Capsicum chinense Jacq)rdquo Jour-nal of the Mexican Chemical Society vol 56 no 1 pp 15ndash182012

[14] L A Castro-Concha J Tuyub-Che A Moo-Mukul F AVazquez-Flota and M L Miranda-Ham ldquoAntioxidant capacityand total phenolic content in fruit tissues from accessions ofCapsicum chinense Jacq (Habanero Pepper) at different stagesof ripeningrdquo The Scientific World Journal vol 2014 Article ID809073 5 pages 2014

[15] M G Gutierrez-Carbajal M Monforte-Gonzalez M L deMiranda-Ham G Godoy-Hernandez and F Vazquez-FlotaldquoInduction of capsaicinoid synthesis in Capsicum chinensecell cultures by salicylic acid or methyl jasmonaterdquo BiologiaPlantarum vol 54 no 3 pp 430ndash434 2010

[16] MMonforte-Gonzalez F Medina-Lara G Gutierrez-Carbajaland F Vazquez-Flota ldquoCapsaicinoid quantitation by in situdensitometry of thin layer chromatography platesrdquo Journal ofLiquid Chromatography and Related Technologies vol 30 no 11pp 1697ndash1704 2007

[17] M J Brown and J S Greenwood ldquoInvolvement of the Golgiapparatus in crystalloid protein deposition inRicinus communiscv Hale seedsrdquo Canadian Journal of Botany vol 68 no 11 pp2353ndash2360 1990

[18] V M Martınez-Juarez N Ochoa-Alejo E Lozoya-Gloriaet al ldquoSpecific synthesis of 551015840-dicapsaicin by cell suspensioncultures of Capsicum annuum var annuum (Chili JalapenoChigol ) and their soluble and NaCl -extracted cell wall proteinfractionsrdquo Journal of Agricultural and Food Chemistry vol 52no 4 pp 972ndash979 2004

[19] Y LangH Kisaka R Sugiyama et al ldquoFunctional loss of pAMTresults in biosynthesis of capsinoids capsaicinoid analogs inCapsicum annuum cv CH-19 sweetrdquo Plant Journal vol 59 no6 pp 953ndash961 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


O CH=O

HO

HO

OH

OH

HO

O

O

CoAS

O

O NH

O Capsaicin

COOH

COOH

O

O

Phenylalanine

Cinnamic acid

PAL Valine

BCAT

AMTHO

O

GABA

CS

NH2

NH2

NH2

120572-Ketoisovalerate

H3C

H3C

H3C

8-Methyl-6-nonenoyl-CoA

CH2ndashNH2

Vanillin

vanillylamine

Figure 1 Involvement of nitrogen or nitrogenous metabolites in capsaicin biosynthesis AMT vanillin aminotransferase BCAT branchedamino acid aminotransferase PAL phenylalanine ammonia-lyase

are involved in CAPs synthesis (Figure 1) These are Pheny-lalanine which provides the phenolic moiety a branchedamino acid either valine or leucine that contributes with thelateral aliphatic chain and 120574-aminobutyric acid (GABA) theamino donor required to form vanillylamine from vanillin([12] Figure 1) Hence as expected CAP contents in pepperpods respond to external nitrogen availability In fact a posi-tive correlation between nitrate and CAPs contents occurs inplacentas from Habanero pepper pods [11] To further assessthe dependence of CAP biosynthesis on nitrogen availabilityisolated Habanero pepper placentas were cultured in vitroand exposed to different nitrate doses Previous to nitrogentreatments placentas were immobilized in calcium alginatein order to keep tissue integrity and metabolic functionalitywas evaluated

2 Materials and Methods

21 Biological Materials Placentas were obtained from podsof an orange accession of Habanero pepper (Capsicumchinense Jacq) collected from commercial plantations in

Yucatan Mexico [13 14] Cell suspensions ofC chinenseweremaintained at the same conditions described previously [15]

22 Tissue Immobilization Process Immature pods (16ndash20days after anthesis ca 3times2 cm longtimeswide)were used shortlyafter harvest Pods were disinfested with ethanol (80) andcommercial bleach (50 equivalent to 3mgL of sodiumhypochlorite) before placentas were excised with scalpeland tweezers Placentas were kept in sterile water prior toimmobilization or transfer to the culturemedium Sections ofplacental tissue (025 cm2) were suspended in a beaker con-taining 25 sodium alginate (Sigma) at room temperatureinside a flow cabinet with constant and gentle stirring in orderto distribute them homogenously in the vessel To obtain thealginate beads a blunt-tip 10mL glass pipette was used todrop ca 1mL of this suspension into cold 1 CaCl

2under

constant gentle stirring Immobilized placentas were allowedto stand for 90 minutes at 4∘C washed with cold distilledwater to eliminate excess calcium and transferred to 250mLErlenmeyer flasks containing 40mL of Murashige and Skoog(MS) mediumwithout growth regulators Each flask received


(a) (b) (c)




4NO3and KNO

3 accounting for a




3 Any side effects





2 and







Time (d)0 7 14 21 28

0

250

500

750

1000

1250


Tota

l sug

ars (120583

g gminus1

DW

)

(a)

Time (d)0 7 14 21 28

0

200

400

600


Tota

l am

ino

acid

s (120583

g gminus1

DW

)

(b)




Time (d)0 7 14 21 28

0

200

400

600

800

1000

1200

1400


Caps

aici

noid

s (120583

g gminus1

DW

)




Time (d)0 7 14 21 28

0

20

40

60

80

100

120

PAL

activ

ity(120583

g ci

nnam

ic ac

idm

inminus1

mg

prot

minus1)


Time (d)0 7 14

0

300

600

900

1200

1500

1800

Pepper placenta40mM

60mM80mM

Caps

aici

noid

s (120583

g gminus1

DW

)



Time (days)

0

20

40

60

80

100

0 7 14 21

80mM

60mM20mM

Caps

aici

n (120583

g gminus1

DW

)

40mM(control)








Acknowledgments


References




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)




4NO3and KNO

3 accounting for a




3 Any side effects





2 and







Time (d)0 7 14 21 28

0

250

500

750

1000

1250


Tota

l sug

ars (120583

g gminus1

DW

)

(a)

Time (d)0 7 14 21 28

0

200

400

600


Tota

l am

ino

acid

s (120583

g gminus1

DW

)

(b)




Time (d)0 7 14 21 28

0

200

400

600

800

1000

1200

1400


Caps

aici

noid

s (120583

g gminus1

DW

)




Time (d)0 7 14 21 28

0

20

40

60

80

100

120

PAL

activ

ity(120583

g ci

nnam

ic ac

idm

inminus1

mg

prot

minus1)


Time (d)0 7 14

0

300

600

900

1200

1500

1800

Pepper placenta40mM

60mM80mM

Caps

aici

noid

s (120583

g gminus1

DW

)



Time (days)

0

20

40

60

80

100

0 7 14 21

80mM

60mM20mM

Caps

aici

n (120583

g gminus1

DW

)

40mM(control)








Acknowledgments


References




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Time (d)0 7 14 21 28

0

250

500

750

1000

1250


Tota

l sug

ars (120583

g gminus1

DW

)

(a)

Time (d)0 7 14 21 28

0

200

400

600


Tota

l am

ino

acid

s (120583

g gminus1

DW

)

(b)




Time (d)0 7 14 21 28

0

200

400

600

800

1000

1200

1400


Caps

aici

noid

s (120583

g gminus1

DW

)




Time (d)0 7 14 21 28

0

20

40

60

80

100

120

PAL

activ

ity(120583

g ci

nnam

ic ac

idm

inminus1

mg

prot

minus1)


Time (d)0 7 14

0

300

600

900

1200

1500

1800

Pepper placenta40mM

60mM80mM

Caps

aici

noid

s (120583

g gminus1

DW

)



Time (days)

0

20

40

60

80

100

0 7 14 21

80mM

60mM20mM

Caps

aici

n (120583

g gminus1

DW

)

40mM(control)








Acknowledgments


References




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Time (d)0 7 14 21 28

0

20

40

60

80

100

120

PAL

activ

ity(120583

g ci

nnam

ic ac

idm

inminus1

mg

prot

minus1)


Time (d)0 7 14

0

300

600

900

1200

1500

1800

Pepper placenta40mM

60mM80mM

Caps

aici

noid

s (120583

g gminus1

DW

)



Time (days)

0

20

40

60

80

100

0 7 14 21

80mM

60mM20mM

Caps

aici

n (120583

g gminus1

DW

)

40mM(control)








Acknowledgments


References




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgments


References




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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