Research ArticleDifference between Burley Tobacco and Flue-CuredTobacco in Nitrate Accumulation and Chemical Regulation ofNitrate and TSNA Contents

Yafei Li1 Hongzhi Shi1 Huijuan Yang1 Jun Zhou2 JingWang1

Ruoshi Bai2 and Dongya Xu1

1Henan Agricultural University National Tobacco Cultivation amp Physiology amp Biochemistry Research CenterZhengzhou 450002 China2Beijing Cigarette Factory of Shanghai Tobacco Group Beijing 100024 China

Correspondence should be addressed to Hongzhi Shi zhihongshi163com

Received 11 July 2017 Accepted 6 November 2017 Published 6 December 2017

Academic Editor Davide Vione

Copyright copy 2017 Yafei Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Tobacco-specific nitrosamines (TSNAs) are harmful carcinogens with nitrate as a precursor of their formation Nitrate content isconsiderably higher in burley tobacco than in flue-cured tobacco but little has been reported on the differences between typesof nitrate accumulation during development We explored nitrate accumulation prior to harvest and examined the effects ofregulatory substances aimed at decreasing nitrate and TSNA accumulation In growth experiments nitrate accumulation in burleyand flue-cured tobacco initially increased but then declined with the highest nitrate content observed during a fast-growth periodWhen treating tobacco crops with molybdenum (Mo) during fast growth nitrate reductase activity in burley tobacco increasedsignificantly but the NO3-N content decreasedThese treatments also yielded significant reductions in NO3-N and TSNA contentsTherefore we suggest that treatment with Mo during the fast-growth period and a Mo-Gfo (Mo-glufosinate) combination at thematurity stage is an effective strategy for decreasing nitrate and TSNAs during cultivation

1 Introduction

Eight types of tobacco-specific nitrosamines (TSNAs) arepresent in tobacco with the majority known to causemalignant tumors in mice rats and hamsters [1 2]N1015840-Nitrosonornicotine (NNN) 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) N1015840-nitrosoanabasine (NAB)and N1015840-nitrosoanatabine (NAT) are key TSNAs with NNNand NNK classified as group 1 carcinogens by the Interna-tional Agency for Research on Cancer [3] The formationof TSNAs during the curing process can be affected by theconcentrations of their nitrate and alkaloid precursors intobacco [4] High temperature and humidity in air-curingbarns or high moisture in tobacco can significantly promoteTSNA formation Good ventilation in burley curing barnsand improved storage conditions contribute to decreasedTSNA formation [5] We have previously found that TSNAsin cured tobacco may greatly increase with exogenous nitrate

application during storage [6] Therefore reducing nitrateaccumulation has become a research focus for decreasingTSNA formation

Nitrate (NO3minus) is one of themajor nitrogen sources taken

up by plants [7 8] which can lead to accumulation in cellvacuoles if it is not reduced reutilized or transported intocytoplasm [9 10] If consumed nitrate is harmful to thehuman body Nitrate can be reduced to nitrite which isreoxidized to nitrate by oxyhemoglobin in the bloodstreamresulting in the formation of methemoglobin and impairingthe capacity of blood to deliver oxygen to body tissues [11ndash14]This condition is referred to as methemoglobinemia and it isharmful to older children and adults Nitrate is also one of themain precursors contributing to formation and accumulationof TSNAs [4] Nitrate is present at concentrations tens tohundreds of times higher in burley tobacco than in flue-curedtobacco with the reasons for this accumulation unclear

HindawiJournal of ChemistryVolume 2017 Article ID 4357456 13 pageshttpsdoiorg10115520174357456

2 Journal of Chemistry

Many factors such as nitrogen management soil fertil-ity tobacco types and varieties and cultivation conditionsare related to nitrate accumulation [4] Increased nitrogenapplication generally gives rise to higher levels of nitrateand low nitrogen efficiency tobacco varieties usually havehigher nitrate accumulation than high-efficiency varietiesunder the same soil nitrogen level [15 16] Differencesin nitrate accumulation among varieties are mainly dueto their differential capacities in absorbing reducing andassimilating nitrate [16ndash19] with high assimilation regardedas a main contributor to low nitrate concentration in thelamina [19 20] Enzymes such as nitrate reductase (NR)and glutamine synthetase (GS) are important in nitrogenmetabolism and their activities have significant effects onnitrate accumulation in tobacco The molybdenum (Mo)cofactor is part of NR composition [20] and symptoms ofMo deficiency and N deficiency are similar in plants [21]Mo application for seed priming and foliar spray is a methodwidely used to enhance crop productivity [22] and is effectivein increasing the relative chlorophyll index plant height leafarea index dry matter production and crop yield [23 24]However there has been little research into the application ofMo to decrease nitrate and TSNA accumulation Glufosinate(Gfo) is a low-residue and effective herbicide in agriculturecultivation known to inhibit glutamine synthetase activity(GSA) and lead to ammonium accumulation as well as theinhibition of photorespiration and photosynthesis in plants[25ndash31] Some investigators have reported that glufosinatemay inhibit the growth of bacteria [32] which may promoteTSNA formation during the tobacco curing stage [4] Variousdoses of Gfo herbicide produce different responses inhibitingGSA in plants with some investigators reporting that spray-ing a suitable amount of Gfo could improve maturity qualityin tobacco [33] However there is little information regardingspraying Gfo to decrease TSNAs in tobacco cultivation

The objective of the present study was to explore char-acteristics of nitrate accumulation in both burley and flue-cured tobacco and compare the differences between types innitrate reductase activity (NRA) and NRAnitrogen applica-tion (NA) to develop strategies for their regulation duringcultivation A field experiment using chemical regulation wasconducted to decrease nitrate and TSNA concentrations influe-cured tobacco and the effects of spraying regulated sub-stances on burley varieties TN86 and TN90 were analyzed todetermine an effectivemethod for reducing nitrate andTSNAconcentrations in burley tobacco The effects of spraying Moduring the fast-growth period and at the maturity stage andof spraying Mo and Gfo together at maturity on NRA GSAammonia volatilization rate (AVR) soluble protein content(SPRO) TSNAs and TSNA precursors were determined

2 Methods

21 Experiment 1 Growth Experiments of Burley and Flue-Cured Tobacco Field and pot experiments were conductedin 2015 in Henan China (33∘151015840521410158401015840N 112∘551015840285110158401015840E)using two tobacco types to explore nitrate accumulation intobacco Two burley tobacco genotypes TN86 and KT204

and two cultivars of flue-cured tobacco honghuadajinyuan(HD) and yunyan 87 (Y87) were used Mean temperatureand precipitation in this region were 241∘C and 510mmrespectively during tobacco cultivation season (from May toSeptember each year)

211 Field Experiments The soil in the field was mainlyyellow loamy soil Soil properties were tested at a depth of0ndash30 cm before transplanting and consisted of an organicmatter content of 1355 g kgminus1 available N of 5501mg kgminus1available K of 12063mg kgminus1 and available P of 1821mg kgminus1and a pH of 713 Nitrogen application was 45 kg haminus2 and180 kg haminus2 for flue-cured tobacco and burley tobacco respec-tively Plants were placed at a density of one plant per0605m2 (column and line spacing per plant 055 times 110mresp) in field experiments Tobacco seedlings were trans-planted to the field on May 1 2015 Burley tobacco was cutonce on July 17 2015 and flue-cured tobacco was picked 3ndash5times beginning on July 12 at 7ndash9-day intervals Experimentaltreatments consisted of a randomized block design with threereplicates Leaf biomass was collected at 30 45 60 and 75days after transplantation (DAT) in field-grown plants withthe final samples picked just prior to harvest Fresh leaveswere fixed for 20min at 105∘C and then dried for 48 h at60∘C NRA and NO3-N contents in leaf were determinedat 30 45 60 and 75 DAT in field

212 Pot Experiments For the pot experiments the soil wasmainly yellow loamy soil Soil was tested at a depth of 0ndash30 cmbefore transplanting andwas similar to that of the field exper-iments with an organic matter content of 1355 g kgminus1 avail-able N of 5510mg kgminus1 available K of 12076mg kgminus1 avail-able P of 1820mg kgminus1 and a pH of 713 Nitrogen applicationwas 45 kg haminus2 and 180 kg haminus2 for flue-cured tobacco andburley tobacco respectively Plants were placed at a densityof one plant per 0605m2 (column and line spacing per plant055 times 110m resp) and transplanted to pots with a 50 cmouter diameter 425 cm inner diameter and 33 cm height andwere buried to a depth of 20ndash25 cm on May 15 2015 Leafbiomass was collected after transplantation at 15 30 45 and60 DAT with the final samples picked just before harvestNRA and NO3-N contents in the leaves were determinedat 15 30 45 and 60 DAT

22 Experiment 2 Nitrate Regulation of Flue-Cured TobaccoUsing Chemical Treatments Nitrate regulation experimentswere conducted in 2014 (Yunnan China 25∘211015840173710158401015840N100∘28101584067510158401015840E) and 2015 (Henan China 33∘151015840521410158401015840N112∘551015840285110158401015840E) using flue-cured tobacco (HD)

221 Soil Property Experiments in Yunnan in 2014 The soilin which the plants were grown was mainly paddy soil witha mean temperature and precipitation of 185∘C and 625mmrespectively during tobacco cultivation season from May toSeptember each year Soil properties were tested at a depth of0ndash20 cm prior to transplantation and had an organic mattercontent of 224 g kgminus1 available N of 12001mg kgminus1 availableK of 15463mg kgminus1 P of 284mg kgminus1 and pH of 648

Journal of Chemistry 3

222 Soil Property Experiments in Henan in 2015 Field soilwas mainly yellow loamy soil Annual mean temperatureand precipitation in this region were 241∘C and 510mmrespectively during tobacco cultivation season from May toSeptember each year Soil properties were tested at a depthof 0ndash30 cm before transplanting and had an organic mattercontent of 1355 g kgminus1 available N of 5501mg kgminus1 availableK of 12063mg kgminus1 and available P of 1821mg kgminus1 and pHof 713 Nitrogen applications were 75 kg haminus2 and 45 kg haminus2in 2014 and 2015 respectively Tobacco seedlings were trans-planted onMay 7 2014 andMay 1 2015 Spraying during fast-growth periods or at the stage of maturity was carried out onJune 17 and July 15 2014 and June 11 and July 10 2015 respec-tively TSNAs NO3-N NO2-N and alkaloids in the tobaccowere determined after curing Field management was carriedout according to conventional practice

The following treatments were applied

(1) A control treatment wherein water only was sprayedduring the fast-growth and maturity stages (CK)

(2) Sodium molybdate sprayed during the fast-growthperiod (FG-Mo)

(3) Sodium molybdate sprayed during the fast-growthperiod and Gfo sprayed at the stage of maturity (M-Gfo)

(4) Sodium molybdate sprayed during the fast-growthperiod and sodium molybdate combined with Gfosprayed at the maturity stage (M-Mo + Gfo)

Sodiummolybdate (Mo) and Gfo doses were determinedin preliminary tests and 10mg Lminus1 Gfo (vv) and 05 (mm)Mo were screened out to spray in field experiments Thedose of Gfo sprayed on tobacco (001 kg hmminus2) was muchlower than its use as a herbicide during agriculture cultivation(040 kg hmminus2 used to control annual weeds and 1-2 kg hmminus2used to control perennial weeds) [34] Residual Gfo in leaveswas low with remaining Gfo decreasing by 15 three daysafter spraying [35]

23 Experiment 3 Nitrate Regulation of Burley Tobacco UsingChemical Treatments Nitrate regulation experiments onburley tobacco (TN86 and KT204 varieties) were conductedin 2015 in Henan China (33∘151015840521410158401015840N 112∘551015840285110158401015840E) Soilconditions treatments and management were as describedin experiment 2 NRA and NO3-N content were determinedfive days after spraying during the fast-growth period NRAGSA NO3-N and SPRO were determined at the seventh dayafter spraying and ammonia volatilization was measured forone full 24 h period from 0800 to 0800 on the seventh dayafter spraying at the stage of maturity AVR was calculated asthe ratio of the amount of ammonia volatilization over timeTSNAs NO3-N NO2-N and alkaloids in the tobacco weredetermined after curing

The length of the various stages of tobacco developmentis as follows [36 37] (1) recovery (adaptation) 30ndash35 days(2) budding (knee-high fast growth and elongation) 20ndash30days (3) maturity (flowering and topping beginning ofharvest and seed formation) 45ndash60 days

24 Chemical Characterization of Soil Soil pH was deter-mined in 1 25 (vv) soilwater suspension organic mattercontent was determined using the potassium bichromatetitrimetricmethod available nitrogenwasmeasured by usingthe alkaline hydrolysis diffusionmethod available potassiumwas measured using the neutral ammonium acetate extrac-tionmethod and available phosphoruswas determined usingalkaline sodium bicarbonate as the extractant in a 20 1 ratio[38]

25 Measurement of NRA GSA SPRO and AVR Tobaccoleaves were sampled at 1000ndash1100 am on sunny daysSamples were frozen and fresh leaves without veins werecut into 2 times 5mm pieces before measurement NRA wasmeasured based on themethod described by Li [39] GSAwasdetermined as per OrsquoNeal and Joy [40] SPRO was assayedaccording to Li [39] AVR was determined by the methodusing airtight equipment [41 42]

26 Measurement of Total Nitrogen (TN) Content NO3-N NO2-N TSNAs and Alkaloids Tobacco samples werelyophilized ground and sieved through a 025mm screenprior to measurement TN was determined using methodsmodified from theChinese Tobacco Industry standard (YCT161159-2002) Samples of 01 g powder mixture containing01 g CuSO4 and 1 g K2SO4 were mixed with 5mL of con-centrated H2SO4 (983mm) in a 50mL digestion tubeand held for 1-2 h at room temperature Samples were thenwarmed to 150∘C for 30min 250∘C for 30min and 370∘C for2 h in a furnace (DS53-380 CIF USA) After cooling approx-imately 10mL deionized water was added and samples wereshaken thoroughly Sample mixtures were cooled for 1-2 hand water was added to maintain the overall volume of thesamples The mixtures were then cooled for 1 h and filteredTN in the supernatant was determined using flow-injection-analysis (AA3 Bran + Luebbe Germany)

NO3-N andNO2-Nwere quantified according to Crutch-field and Grove [43] The individual alkaloids were analyzedusing a gas chromatograph as described by Jack and Bush[43] Methyl tert-butyl ether was applied as the extractionsolvent with N-hexadecane according to internal standards[44] NNN NNK NAT and NAB contents were determinedaccording to SPE-LC-MSMS methods [45ndash47] The totalTSNA concentration was calculated by summing the NNNNNK NAT and NAB [6]

27 Statistical Analyses Comparisons were made using anal-yses of variance (ANOVAs) and least significant differencesfor NRA GSA AVR NO119883 alkaloids and TSNAs with 119901 lt005 considered significant based on three replicates Datawere analyzed in Statistical Package for the Social Sciences(SPSS 200) and figures were created using Origin 90Pearson correlations were used to analyze the relationshipsbetween TSNAs and their precursors

3 Results and Discussion

31 Features of NO3-N Content and NRA in Flue-CuredTobacco and Burley Tobacco In field and pot experimentsnitrate content in both burley tobacco and flue-cured tobacco

4 Journal of Chemistry

Table 1 ANOVA results of the effects of chemical regulation year and tobacco variety and their interactions on LDMandDMbefore harvest

Year Treatment LDM (gplant) DM (gplant) Variety Treatment LDM (gplant) DM (gplant)

2014

CK 13070 plusmn 733b 24070 plusmn 791a

KT204

CK 12413 plusmn 326ab 24413 plusmn 442a

FG-Mo 14935 plusmn 349a 26035 plusmn 378a FG-Mo 13405 plusmn 352a 25605 plusmn 468a

M-Gfo 11433 plusmn 275c 22433 plusmn 289b M-Gfo 11849 plusmn 226b 23749 plusmn 441a

M-Mo + Gfo 13785 plusmn 278ab 24785 plusmn 335a M-Mo + Gfo 12817 plusmn 315ab 24817 plusmn 430a

2015

CK 14270 plusmn 584a 25570 plusmn 755a

TN86

CK 11341 plusmn 454ab 21541 plusmn 569ab

FG-Mo 15527 plusmn 499a 26860 plusmn 701a FG-Mo 12534 plusmn 292a 23301 plusmn 437a

M-Gfo 14102 plusmn 430a 25402 plusmn 603a M-Gfo 10413 plusmn 338b 20413 plusmn 453b

M-Mo + Gfo 15152 plusmn 720a 26452 plusmn 893a M-Mo + Gfo 11665 plusmn 328ab 22065 plusmn 558ab

Year (Y) 202lowastlowast 065lowastlowast Variety (V) 090lowast 194lowastlowast

Treatment (T) 471lowastlowast 344lowast Treatment (T) 528lowastlowast 199

Year (Y) times treatment (T) 671lowastlowast 513lowastlowast Variety (V) times treatment (T) 788lowastlowast 1438lowastlowast

Different letters within the same column indicate significant differences among treatments at 119901 lt 005 Symbols lowastlowast and lowast indicate significant difference at 001or 005 respectively

increased over the period of development and presented atrend of ldquorise-fallrdquo prior to harvest (Figure 1) Nitrate contentwas at its highest during the fast-growth period Nitrate isdifficult to recycle once stored in cells [48] Hence avoidingnitrate accumulation during the fast-growth stage may beeffective in reducing nitrate accumulation in cured tobacco

In general the amount of nitrogen fertilizers used onburley tobacco was almost 3ndash5 times higher than that used onflue-cured tobacco but the yield was not significantly differ-ent between them [49] NRA and NO3-N contents betweenburley tobacco and flue-cured tobacco were significantlydifferent with NRANA in flue-cured tobacco significantlyhigher than in burley tobacco During tobacco developmentthe NO3-N content in burley tobacco was higher than that influe-cured tobacco in both field and pot experiments NRAwas readily affected by nitrogen application with nitrogenapplication on burley tobacco 4-fold greater than that in flue-cured tobacco production NRANA in flue-cured tobaccowas higher than in burley tobacco in both field and pot exper-iments In addition weak nitrogen assimilation of burleytobacco may be an important cause of nitrate accumulation[50]

32 Effects of Chemical Regulation on Leaf Biological Yield(LDM) and Above-Ground Dry Matter Weight (DM) LDMand DM were used to evaluate whether plants were growingwell and to predict yield in tobacco cultivation [36] It hasbeen reported thatDM yield and product quality all decreaseunder a Mo-deficient condition [51] In this work LDM andDM increased with Mo being sprayed during the fast-growthperiod which has been shown to dilute nitrate concentration[50] The main effects of chemical treatment and year weresignificantly observed for LDM andDMover the two years ofobservation (119901 lt 005) (Table 1) Variation between tobaccovarieties also significantly affected LDM and DM LDM andDMin tobacco increased underMo treatment during the fast-growth period Meanwhile LDM and DM showed a decreasewith spraying of Gfo at the maturity stage

33 Effects of Chemical Regulation on NRA GSA AVR SPROandNO3-NContent NRA andNO3-N content in both TN86and KT204 exhibited increasing trends (Figure 2) whichwere closely related to the maximum uptake of nutrientsduring the rapid growth stage [36] Additionally enhancingnitrogen assimilation ability and decreasing nitrate storagewere key in reducing nitrate accumulation in tobacco duringthis period Under the Mo treatment during the fast-growthperiod NRA in TN86 and KT204 increased by 157ndash1181and 172ndash1058 respectively but NO3-N content in TN86and KT204 decreased correspondingly by 1016ndash5808 and1004ndash4887 respectively (119901 lt 001)

Composition of tobacco at the stage of maturity is signif-icantly indicative of the components of cured tobacco andimproving chemical composition during this stage is useful inenhancing tobacco quality [52] NR and GS are key enzymesin the process of nitrogen reduction and assimilation inplants and GS plays an important role in the first step ofNH4+ assimilation [53] NRA AVR GSA and SPRO in

burley tobacco were significantly affected by spraying Gfo atthe maturity stage (Figures 3(a)ndash3(h)) Gfo application caninhibit GSA and cause ammonia emissions of almost 10 ofcanopy nitrogen content [26] Compared with CK the GSAand SPRO of Gfo-sprayed tobacco significantly decreasedandAVR significantly increasedHence sprayingMo andGfoat maturity was effective in decreasing nitrate accumulationand promoting nitrogen loss in tobacco (Figure 8)

34 Effects of Chemical Regulation on TSNA PrecursorsNO3-N NO2-N and alkaloids are precursors of TSNAsand decreasing precursors is effective in reducing TSNAformation in tobacco Sufficient NO3-N content can greatlypromote TSNA formation during tobacco storage andreducing NO3-N accumulation is key in decreasing TSNAformation [54] As shown above treatment with Mo andGfo significantly decreased TN NO3-N NO2-N and NO3-NTN but did not affect alkaloid levels in burley tobacco(Figures 4(a)ndash4(j)) Spraying Mo during periods of fast

Journal of Chemistry 5

Field experiment

807570656055504540353025

Days aer transplantation (d)

0

10

20

30

NRA

NA

40

50

60

70

HDY87

KT204

TN86

(a)

Pot experiment

656055504540353025201510

Days aer transplantation (d)

0

10

20NRA

NA

30

40

50

60

HDY87

KT204

TN86

(b)

Field experiment

807570656055504540353025

Days aer transplantation (d)

50

100

150

200

250

300

NRA

(g

mＡminus

1Ｂminus1

FW)

HDY87

KT204

TN86

(c)

Pot experiment

656055504540353025201510

Days aer transplantation (d)

50

75

100

125

150

175

200N

RA (

g mＡminus

1Ｂminus1

FW)

HDY87

KT204

TN86

(d)

Field experiment

N

3-N

cont

ent (

gＡminus

1D

W) 5000

4000

3000

2000

1000

075604530 Before

Days aer transplantation (d)

HDY87

KT204

TN86

harvest

(e)

Pot experiment

60453015

Days aer transplantation (d)

Beforeharvest

0

1000

2000

3000

4000

5000

N

3-N

cont

ent (

gＡminus

1D

W)

HDY87

KT204

TN86

(f)

Figure 1 Difference between burley tobacco and flue-cured tobacco inNRANRANA andNO3-N content of leaves Burley tobacco varietieswere KT204 and TN86 and flue-cured tobacco varieties were HD and Y87 NA nitrogen application (HD and Y87 45 kg haminus2 KT204 andTN86 180 kg haminus2) NRA nitrate reductase activity Error bars indicate standard error of the means (119899 = 3)

6 Journal of Chemistry

TN86

CKFG-Mo

175

200

225

250

275

300

NRA

(g

mＡminus

1Ｂminus1

FW)

1 d 2 d 3 d 4 d 5 d6 hTime aer spraying

(a)

KT204

CKFG-Mo

200

225

250

275

300

NRA

(g

mＡminus

1Ｂminus1

FW)

1 d 2 d 3 d 4 d 5 d6 hTime aer spraying

(b)

TN86

CKFG-Mo

1600

2400

3200

4000

4800

N

3-N

cont

ent (

gＡminus

1D

W)

2 3 4 51

Time aer spraying (d)

(c)

KT204

CKFG-Mo

1600

2000

2400

2800

3200

3600N

3-N

cont

ent (

gＡminus

1D

W)

2 3 4 51

Time aer spraying (d)

(d)

Figure 2 Changes in the NRA andNO3-N content of burley tobacco underMo treatment during fast-growth period NRA nitrate reductaseactivity NRA in tobacco was determined at 6 h and on days 1ndash5 after spraying Error bars indicate standard error of means (119899 = 3)

growth led to significantly lower NO3-N content in KT204and TN86 Spraying Mo during the fast-growth period andsimultaneously spraying Mo and Gfo at the stage of maturityled to a significant decrease in NO3-N and NO2-N content inKT204 and TN86

35 Effects of Chemical Regulation on TSNAContents Auxinnaphthylacetic acid salicylic acid and malonic acid havebeen previously applied to decrease TSNA formation butthese may affect tobacco development and growth yield orquality [55 56] In this study we aimed to characterize achemical regulation strategy for decreasing TSNA precursors

so as to diminish TSNA formation in tobacco Yearly differ-ences in NO3-N NO2-N and TSNA contents in flue-curedtobacco were significant but TN and alkaloid levels werenot (Table 2) Regulatory treatments significantly affectedTN NO3-N alkaloid level and TSNA concentrations in flue-cured tobacco Varieties of burley tobacco were differentin TN NO3-N NO2-N alkaloid level and TSNA concen-trations and chemical regulation treatments significantlyaffected TN NO3-N and TSNA concentrations

As can be seen in Figure 5 spraying Mo during the fast-growth period and spraying Gfo at the stage of maturitydecreased TSNA concentrations in flue-cured tobacco but

Journal of Chemistry 7

CK

lowast

lowastlowast

lowastlowast

FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(a)

lowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

004

005

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(b)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

AVR

(gＧ

minus2Ｂminus1)

(c)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(d)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(e)

lowastlowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(f)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

40

AVR

(gＧ

minus2Ｂminus1)

(g)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(h)

Figure 3 Effects of Mo and Gfo treatments on NRA AVR GSA and SPRO in burley tobacco Error bars represent standard error (119899 = 3)NRA nitrate reductase activity AVR ammonia volatilization rate GSA glutamine synthetase activity SPRO total soluble protein content(andashd) KT204 (endashh) TN86 Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

8 Journal of Chemistry

Table 2 ANOVA comparison of the effects of regulatory treatments year and tobacco variety and their interactions on TN NO3-N NO2-Nalkaloid and TSNA concentrations in tobacco

Types Effect TN NO3-N NO2-N Alkaloid TSNAs DF

Flue-cured tobaccoYear (Y) 005ns 020lowast 001lowastlowast 160ns 004lowastlowast 1

Treatment (T) 901lowastlowast 1533lowastlowast 021ns 465lowast 719lowastlowast 3Year (Y) times Treatment (T) 854lowastlowast 5561lowastlowast 1371lowastlowast 573lowastlowast 3052lowastlowast 7

Burley tobaccoVariety (V) 097lowast 018lowast 1108lowastlowast 002lowastlowast 118lowast 1

Treatment (T) 359lowast 1934lowastlowast 097ns 028ns 1868lowastlowast 3Variety (V) times Treatment (T) 566lowastlowast 27557lowastlowast 2841lowastlowast 1314lowastlowast 27477lowastlowast 7

119865-values and significance levels are given (lowastlowast119901 lt 001 lowast119901 lt 005 and ns119901 ge 005)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(a)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowastlowastlowast

lowastlowast

0

500

1000

1500

2000

2500

3000

3500

N

3-N

cont

ent (

gＡminus

1D

W)

(b)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowast

0

1

2

3

4

N

2-N

cont

ent (

gＡminus

1D

W)

(c)M

-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1

2

3

4

Alk

aloi

d co

nten

t (

DW

)

(d)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

8

N

3-N

TN

()

(e)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(f)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

500

1000

1500

2000

2500

N

3-N

cont

ent (

gＡminus

1D

W)

(g)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowastlowast

00

05

10

15

20

N

2-N

cont

ent (

gＡminus

1D

W)

(h)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

2

4

6

Alk

aloi

d co

nten

t (

DW

)

(i)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

N

3-N

TN

()

(j)

Figure 4 Effects of treatments on total nitrogen content NO3-N content NO2-N content alkaloid content and NO3-NTN in burleytobacco NO3-NTN ratio of NO3-N and total nitrogen content (TN) Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicatesignificant difference at 001 or 005 respectively

the effect of spraying Mo during the fast-growth period wassignificantly different (119901 lt 005) Spraying Mo during thefast-growth period and spraying Mo and Gfo at maturityproduced the best results on TSNA concentrations among alltreatments in both 2014 and 2015 Spraying Mo during thefast-growth period could significantly reduce concentrations

of NNN NAB andNAT SprayingMo during the fast-growthperiod and spraying Gfo at the stage of maturity decreasedNNNNABNAT and total TSNA concentration in both 2014and 2015 respectively

TSNA accumulation in burley tobacco was much higherthan in flue-cured tobacco However effects of regulatory

Journal of Chemistry 9

2014

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

50

60

70

80

90

100

NN

N (n

gＡminus

1D

W)

(a)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

25

30

35

40

NAT

(ngＡminus

1D

W)

(b)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

20

25

30

35

40

NA

B (n

gＡminus

1D

W)

(c)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowastlowastlowast

lowastlowast

20

30

40

50

NN

K (n

gＡminus

1D

W)

(d)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast lowastlowast

lowastlowast

100

125

150

175

200

TSN

As (

ngＡminus

1D

W)

(e)2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

40

50

60

70

80

90

NN

N (n

gＡminus

1D

W)

(f)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

30

40

50

60

70

80

90

100

NAT

(ngＡminus

1D

W)

(g)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

10

15

20

25

30

NA

B (n

gＡminus

1D

W)

(h)

2015

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK20

25

30

35

40

45

50

55

60

NN

K (n

gＡminus

1D

W)

(i)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

80

120

160

200

240

TSN

As (

ngＡminus

1D

W)(j)

Figure 5 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in flue-cured tobacco Error bars indicatestandard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

treatment were more pronounced in burley tobacco Withinburley varieties the TSNA concentrations in KT204 werehigher than that in TN86 (Figures 6(a)ndash6(e)) Spraying ofModuring the fast-growth period led to significantly lowerNNNNAB and total TSNA concentrations in KT204 Sprayingof Gfo at maturity led to significant decreases in NNNNAT NNK and TSNA concentrations in TN86 The TSNA-regulating effects of the two treatments were optimized byspraying Mo during the fast-growth period and Gfo at thestage of maturity NNN NAT NAB NNK and total TSNAconcentration decreased in KT204 and TN86

36 Correlation Analysis Linear relationships betweenTSNAs alkaloids and NO3-N were significantly different(Figures 7(a)ndash7(c)) Total TSNA concentration in tobaccoincreased with increasing alkaloid and NO3-N contentespecially in burley tobacco The positive correlations

between TSNAs and their precursors were also reported byLewis et al [57] who suggested that NO3-N was a strongercontributing factor to higher TSNA levels than increasedalkaloid levels in burley tobacco

4 Conclusion

Nitrate was higher in burley tobacco than in flue-curedtobacco with both types showing peak nitrate content duringthe fast-growth period Under Mo treatment at the stage ofmaturity to avoid nitrate accumulation NRA LDM and DMin tobacco leaves increased SprayingMo in combinationwithGfo at the stage of maturity led to increased NRA and lowerGSA in tobacco which could help decrease nitrate and nitritecontent by increasing nitrogen loss via ammonia volatiliza-tion In summary spraying Mo during fast growth andspraying Mo with Gfo at the stage of maturity were effectivein reducing the formation of TSNAs

10 Journal of Chemistry

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

500

1000

1500

2000

NN

N (n

gＡminus

1D

W)

(a)

lowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

10

20

30

NA

B (n

gＡminus

1D

W)

(b)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

200

400

600

NAT

(ngＡminus

1D

W)

(c)

lowastlowastlowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(d)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1000

2000

3000

TSN

As (

ngＡminus

1D

W)

(e)

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

NN

N (n

gＡminus

1D

W)

(f)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

10

20

30

NA

B (n

gＡminus

1D

W)

(g)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

200

400

600

800N

AT (n

gＡminus

1D

W)

(h)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(i)

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

4000

TSN

As (

ngＡminus

1D

W)

(j)

Figure 6 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in burley tobacco varieties TN86 andKT204 Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

Burley tobacco

Flue-cured tobacco

R2 = 07220lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

3 4

Alkaloid content ( DW)

y = 15176x minus 35891

(a)

Burley tobacco

Flue-cured tobacco

R2 = 00557

0

2000

4000

TSN

As (

ngＡminus

1D

W)

321

N2-N content (g Ａminus1 DW)

y = 48834x + 17464

(b)

N3-N content (g Ａminus1 DW)3000200010000

Burley tobacco

Flue-cured tobacco

R2 = 07983lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

y = 121643x minus 28745602

(c)

Figure 7 Correlation analysis between TSNAs alkaloid NO2-N and NO3-N in tobacco Symbol lowastlowast indicates significant correlation at119901 lt 001

Journal of Chemistry 11

Gln

NR NiR

Glu Glu

Protein

Gln 2OG

NR NiRGS

TCA

TSNAs

Mo

NR Protein

NR GS

In fastgrowthperiod

At maturitystage

Leaf

Leaf

Nitratestorage

Nitrateaccumulation

Leaf

Nitratetransport

Glu Glu

Leaf

2OG

N（3

N（3

N（3

N3-N

N3-N

N3-N

N2-N

N2-N

Nx + lkaloid

MＩ + fo

Figure 8 Mechanisms for decreasing nitrate and TSNA concentrations in tobacco by spraying regulating chemicals Gfo glufosinate NRnitrate reductase NRA nitrate reductase activity NiR nitrite reductase GS glutamine synthetase GSA glutamine synthetase activity Glnglutamine Glu glutamate and OG oxaloacetate After sprayingMo on tobacco during the fast-growth period nitrate significantly decreasedwhile NRA and soluble protein content increasedThese decreased the amount of nitrate storage and promoted tobacco development duringthe fast-growth period After spraying Mo during the fast-growth stage and spraying Mo and Gfo at the stage of maturity NRA increasedand GSA decreased in tobacco which can significantly reduce nitrate accumulation and TSNA formation by nitrogen loss due to ammoniavolatilization

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank Editage (httpswwweditagecom) forEnglish language editing and publication support

References

[1] H R Burton N K Dye and L P Bush ldquoRelationship betweentobacco-specific nitrosamines and nitrite from different air-cured tobacco varietiesrdquo Journal of Agricultural and FoodChemistry vol 42 no 9 pp 2007ndash2011 1994

[2] B Siminszky L Gavilano S W Bowen and R E DeweyldquoConversion of nicotine to nornicotine in Nicotiana tabacumis mediated by CYP82E4 a cytochrome P450 monooxygenaserdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 41 pp 14919ndash14924 2005

[3] IARC ldquoSmokeless tobacco and some tobacco-specific N-nitrosamines monographs on the evaluation of carcinogenicrisks to humansrdquo National Publishing Group vol 89 Article ID1e592 2005

[4] H Shi R Wang and L P Bush ldquoThe relationships betweenTSNAs and their precursors in burley tobacco from differentregions and varieties JournalofFoodrdquoAgricultureEnvironmentvol 10 no 2 pp 1048ndash1052 2012

[5] H R Burton G H Childs Jr R A Andersen and P D Flem-ing ldquoChanges in chemical composition of Burley tobacco dur-ing senescence and curing 3 Tobacco-specific nitrosaminesrdquoJournal of Agricultural and Food Chemistry vol 37 no 2 pp426ndash430 1989

[6] H Shi R Wang L P Bush et al ldquoChanges in TSNA Contentsduring Tobacco Storage and the Effect of Temperature andNitrate Level on TSNA Formationrdquo Journal of Agricultural andFood Chemistry vol 61 pp 11588ndash11594 2013

[7] K Fytianos and P Zarogiannis ldquoNitrate and nitrite accumula-tion in fresh vegetables from Greecerdquo Bulletin of EnvironmentalContamination and Toxicology vol 62 no 2 pp 187ndash192 1999

[8] Y Y Wang P K Hsu and Y F Tsay ldquoUptake allocation andsignaling of nitraterdquo Trends in Plant Science vol 17 no 8 pp458ndash467 2012

[9] K S Reddy and R C Menary ldquoNitrate reductase and nitrateaccumulation in relation to nitrate toxicity in Boronia meg-astigmardquo Physiologia Plantarum vol 78 no 3 pp 430ndash4341990

[10] Y Han Q Liao Y Yu et al ldquoNitrate reutilization mechanismsin the tonoplast of two Brassica napus genotypes with differentnitrogen use efficiencyrdquoActa Physiologiae Plantarum vol 37 no2 2015

[11] H Kosaka K Imaizumi K Imai and I Tyuma ldquoStoichiometryof the reaction of oxyhemoglobin with nitriterdquo BBA - ProteinStructure vol 581 no 1 pp 184ndash188 1979

[12] P Santamaria ldquoNitrate in vegetables toxicity content intakeand EC regulationrdquo Journal of the Science of Food and Agricul-ture vol 86 no 1 pp 10ndash17 2006

[13] A T Diplock P J Aggett M Ashwell F Bornet E B FernandM B Roberfroid ldquoScientific concepts of functional foods in

Journal of Chemistry 3

222 Soil Property Experiments in Henan in 2015 Field soilwas mainly yellow loamy soil Annual mean temperatureand precipitation in this region were 241∘C and 510mmrespectively during tobacco cultivation season from May toSeptember each year Soil properties were tested at a depthof 0ndash30 cm before transplanting and had an organic mattercontent of 1355 g kgminus1 available N of 5501mg kgminus1 availableK of 12063mg kgminus1 and available P of 1821mg kgminus1 and pHof 713 Nitrogen applications were 75 kg haminus2 and 45 kg haminus2in 2014 and 2015 respectively Tobacco seedlings were trans-planted onMay 7 2014 andMay 1 2015 Spraying during fast-growth periods or at the stage of maturity was carried out onJune 17 and July 15 2014 and June 11 and July 10 2015 respec-tively TSNAs NO3-N NO2-N and alkaloids in the tobaccowere determined after curing Field management was carriedout according to conventional practice

The following treatments were applied

(1) A control treatment wherein water only was sprayedduring the fast-growth and maturity stages (CK)

(2) Sodium molybdate sprayed during the fast-growthperiod (FG-Mo)

(3) Sodium molybdate sprayed during the fast-growthperiod and Gfo sprayed at the stage of maturity (M-Gfo)

(4) Sodium molybdate sprayed during the fast-growthperiod and sodium molybdate combined with Gfosprayed at the maturity stage (M-Mo + Gfo)

Sodiummolybdate (Mo) and Gfo doses were determinedin preliminary tests and 10mg Lminus1 Gfo (vv) and 05 (mm)Mo were screened out to spray in field experiments Thedose of Gfo sprayed on tobacco (001 kg hmminus2) was muchlower than its use as a herbicide during agriculture cultivation(040 kg hmminus2 used to control annual weeds and 1-2 kg hmminus2used to control perennial weeds) [34] Residual Gfo in leaveswas low with remaining Gfo decreasing by 15 three daysafter spraying [35]

23 Experiment 3 Nitrate Regulation of Burley Tobacco UsingChemical Treatments Nitrate regulation experiments onburley tobacco (TN86 and KT204 varieties) were conductedin 2015 in Henan China (33∘151015840521410158401015840N 112∘551015840285110158401015840E) Soilconditions treatments and management were as describedin experiment 2 NRA and NO3-N content were determinedfive days after spraying during the fast-growth period NRAGSA NO3-N and SPRO were determined at the seventh dayafter spraying and ammonia volatilization was measured forone full 24 h period from 0800 to 0800 on the seventh dayafter spraying at the stage of maturity AVR was calculated asthe ratio of the amount of ammonia volatilization over timeTSNAs NO3-N NO2-N and alkaloids in the tobacco weredetermined after curing

The length of the various stages of tobacco developmentis as follows [36 37] (1) recovery (adaptation) 30ndash35 days(2) budding (knee-high fast growth and elongation) 20ndash30days (3) maturity (flowering and topping beginning ofharvest and seed formation) 45ndash60 days

24 Chemical Characterization of Soil Soil pH was deter-mined in 1 25 (vv) soilwater suspension organic mattercontent was determined using the potassium bichromatetitrimetricmethod available nitrogenwasmeasured by usingthe alkaline hydrolysis diffusionmethod available potassiumwas measured using the neutral ammonium acetate extrac-tionmethod and available phosphoruswas determined usingalkaline sodium bicarbonate as the extractant in a 20 1 ratio[38]

25 Measurement of NRA GSA SPRO and AVR Tobaccoleaves were sampled at 1000ndash1100 am on sunny daysSamples were frozen and fresh leaves without veins werecut into 2 times 5mm pieces before measurement NRA wasmeasured based on themethod described by Li [39] GSAwasdetermined as per OrsquoNeal and Joy [40] SPRO was assayedaccording to Li [39] AVR was determined by the methodusing airtight equipment [41 42]

26 Measurement of Total Nitrogen (TN) Content NO3-N NO2-N TSNAs and Alkaloids Tobacco samples werelyophilized ground and sieved through a 025mm screenprior to measurement TN was determined using methodsmodified from theChinese Tobacco Industry standard (YCT161159-2002) Samples of 01 g powder mixture containing01 g CuSO4 and 1 g K2SO4 were mixed with 5mL of con-centrated H2SO4 (983mm) in a 50mL digestion tubeand held for 1-2 h at room temperature Samples were thenwarmed to 150∘C for 30min 250∘C for 30min and 370∘C for2 h in a furnace (DS53-380 CIF USA) After cooling approx-imately 10mL deionized water was added and samples wereshaken thoroughly Sample mixtures were cooled for 1-2 hand water was added to maintain the overall volume of thesamples The mixtures were then cooled for 1 h and filteredTN in the supernatant was determined using flow-injection-analysis (AA3 Bran + Luebbe Germany)

NO3-N andNO2-Nwere quantified according to Crutch-field and Grove [43] The individual alkaloids were analyzedusing a gas chromatograph as described by Jack and Bush[43] Methyl tert-butyl ether was applied as the extractionsolvent with N-hexadecane according to internal standards[44] NNN NNK NAT and NAB contents were determinedaccording to SPE-LC-MSMS methods [45ndash47] The totalTSNA concentration was calculated by summing the NNNNNK NAT and NAB [6]

27 Statistical Analyses Comparisons were made using anal-yses of variance (ANOVAs) and least significant differencesfor NRA GSA AVR NO119883 alkaloids and TSNAs with 119901 lt005 considered significant based on three replicates Datawere analyzed in Statistical Package for the Social Sciences(SPSS 200) and figures were created using Origin 90Pearson correlations were used to analyze the relationshipsbetween TSNAs and their precursors

3 Results and Discussion

31 Features of NO3-N Content and NRA in Flue-CuredTobacco and Burley Tobacco In field and pot experimentsnitrate content in both burley tobacco and flue-cured tobacco

4 Journal of Chemistry

Table 1 ANOVA results of the effects of chemical regulation year and tobacco variety and their interactions on LDMandDMbefore harvest

Year Treatment LDM (gplant) DM (gplant) Variety Treatment LDM (gplant) DM (gplant)

2014

CK 13070 plusmn 733b 24070 plusmn 791a

KT204

CK 12413 plusmn 326ab 24413 plusmn 442a

FG-Mo 14935 plusmn 349a 26035 plusmn 378a FG-Mo 13405 plusmn 352a 25605 plusmn 468a

M-Gfo 11433 plusmn 275c 22433 plusmn 289b M-Gfo 11849 plusmn 226b 23749 plusmn 441a

M-Mo + Gfo 13785 plusmn 278ab 24785 plusmn 335a M-Mo + Gfo 12817 plusmn 315ab 24817 plusmn 430a

2015

CK 14270 plusmn 584a 25570 plusmn 755a

TN86

CK 11341 plusmn 454ab 21541 plusmn 569ab

FG-Mo 15527 plusmn 499a 26860 plusmn 701a FG-Mo 12534 plusmn 292a 23301 plusmn 437a

M-Gfo 14102 plusmn 430a 25402 plusmn 603a M-Gfo 10413 plusmn 338b 20413 plusmn 453b

M-Mo + Gfo 15152 plusmn 720a 26452 plusmn 893a M-Mo + Gfo 11665 plusmn 328ab 22065 plusmn 558ab

Year (Y) 202lowastlowast 065lowastlowast Variety (V) 090lowast 194lowastlowast

Treatment (T) 471lowastlowast 344lowast Treatment (T) 528lowastlowast 199

Year (Y) times treatment (T) 671lowastlowast 513lowastlowast Variety (V) times treatment (T) 788lowastlowast 1438lowastlowast

Different letters within the same column indicate significant differences among treatments at 119901 lt 005 Symbols lowastlowast and lowast indicate significant difference at 001or 005 respectively

increased over the period of development and presented atrend of ldquorise-fallrdquo prior to harvest (Figure 1) Nitrate contentwas at its highest during the fast-growth period Nitrate isdifficult to recycle once stored in cells [48] Hence avoidingnitrate accumulation during the fast-growth stage may beeffective in reducing nitrate accumulation in cured tobacco

In general the amount of nitrogen fertilizers used onburley tobacco was almost 3ndash5 times higher than that used onflue-cured tobacco but the yield was not significantly differ-ent between them [49] NRA and NO3-N contents betweenburley tobacco and flue-cured tobacco were significantlydifferent with NRANA in flue-cured tobacco significantlyhigher than in burley tobacco During tobacco developmentthe NO3-N content in burley tobacco was higher than that influe-cured tobacco in both field and pot experiments NRAwas readily affected by nitrogen application with nitrogenapplication on burley tobacco 4-fold greater than that in flue-cured tobacco production NRANA in flue-cured tobaccowas higher than in burley tobacco in both field and pot exper-iments In addition weak nitrogen assimilation of burleytobacco may be an important cause of nitrate accumulation[50]

32 Effects of Chemical Regulation on Leaf Biological Yield(LDM) and Above-Ground Dry Matter Weight (DM) LDMand DM were used to evaluate whether plants were growingwell and to predict yield in tobacco cultivation [36] It hasbeen reported thatDM yield and product quality all decreaseunder a Mo-deficient condition [51] In this work LDM andDM increased with Mo being sprayed during the fast-growthperiod which has been shown to dilute nitrate concentration[50] The main effects of chemical treatment and year weresignificantly observed for LDM andDMover the two years ofobservation (119901 lt 005) (Table 1) Variation between tobaccovarieties also significantly affected LDM and DM LDM andDMin tobacco increased underMo treatment during the fast-growth period Meanwhile LDM and DM showed a decreasewith spraying of Gfo at the maturity stage

33 Effects of Chemical Regulation on NRA GSA AVR SPROandNO3-NContent NRA andNO3-N content in both TN86and KT204 exhibited increasing trends (Figure 2) whichwere closely related to the maximum uptake of nutrientsduring the rapid growth stage [36] Additionally enhancingnitrogen assimilation ability and decreasing nitrate storagewere key in reducing nitrate accumulation in tobacco duringthis period Under the Mo treatment during the fast-growthperiod NRA in TN86 and KT204 increased by 157ndash1181and 172ndash1058 respectively but NO3-N content in TN86and KT204 decreased correspondingly by 1016ndash5808 and1004ndash4887 respectively (119901 lt 001)

Composition of tobacco at the stage of maturity is signif-icantly indicative of the components of cured tobacco andimproving chemical composition during this stage is useful inenhancing tobacco quality [52] NR and GS are key enzymesin the process of nitrogen reduction and assimilation inplants and GS plays an important role in the first step ofNH4+ assimilation [53] NRA AVR GSA and SPRO in

burley tobacco were significantly affected by spraying Gfo atthe maturity stage (Figures 3(a)ndash3(h)) Gfo application caninhibit GSA and cause ammonia emissions of almost 10 ofcanopy nitrogen content [26] Compared with CK the GSAand SPRO of Gfo-sprayed tobacco significantly decreasedandAVR significantly increasedHence sprayingMo andGfoat maturity was effective in decreasing nitrate accumulationand promoting nitrogen loss in tobacco (Figure 8)

34 Effects of Chemical Regulation on TSNA PrecursorsNO3-N NO2-N and alkaloids are precursors of TSNAsand decreasing precursors is effective in reducing TSNAformation in tobacco Sufficient NO3-N content can greatlypromote TSNA formation during tobacco storage andreducing NO3-N accumulation is key in decreasing TSNAformation [54] As shown above treatment with Mo andGfo significantly decreased TN NO3-N NO2-N and NO3-NTN but did not affect alkaloid levels in burley tobacco(Figures 4(a)ndash4(j)) Spraying Mo during periods of fast

Journal of Chemistry 5

Field experiment

807570656055504540353025

Days aer transplantation (d)

0

10

20

30

NRA

NA

40

50

60

70

HDY87

KT204

TN86

(a)

Pot experiment

656055504540353025201510

Days aer transplantation (d)

0

10

20NRA

NA

30

40

50

60

HDY87

KT204

TN86

(b)

Field experiment

807570656055504540353025

Days aer transplantation (d)

50

100

150

200

250

300

NRA

(g

mＡminus

1Ｂminus1

FW)

HDY87

KT204

TN86

(c)

Pot experiment

656055504540353025201510

Days aer transplantation (d)

50

75

100

125

150

175

200N

RA (

g mＡminus

1Ｂminus1

FW)

HDY87

KT204

TN86

(d)

Field experiment

N

3-N

cont

ent (

gＡminus

1D

W) 5000

4000

3000

2000

1000

075604530 Before

Days aer transplantation (d)

HDY87

KT204

TN86

harvest

(e)

Pot experiment

60453015

Days aer transplantation (d)

Beforeharvest

0

1000

2000

3000

4000

5000

N

3-N

cont

ent (

gＡminus

1D

W)

HDY87

KT204

TN86

(f)

Figure 1 Difference between burley tobacco and flue-cured tobacco inNRANRANA andNO3-N content of leaves Burley tobacco varietieswere KT204 and TN86 and flue-cured tobacco varieties were HD and Y87 NA nitrogen application (HD and Y87 45 kg haminus2 KT204 andTN86 180 kg haminus2) NRA nitrate reductase activity Error bars indicate standard error of the means (119899 = 3)

6 Journal of Chemistry

TN86

CKFG-Mo

175

200

225

250

275

300

NRA

(g

mＡminus

1Ｂminus1

FW)

1 d 2 d 3 d 4 d 5 d6 hTime aer spraying

(a)

KT204

CKFG-Mo

200

225

250

275

300

NRA

(g

mＡminus

1Ｂminus1

FW)

1 d 2 d 3 d 4 d 5 d6 hTime aer spraying

(b)

TN86

CKFG-Mo

1600

2400

3200

4000

4800

N

3-N

cont

ent (

gＡminus

1D

W)

2 3 4 51

Time aer spraying (d)

(c)

KT204

CKFG-Mo

1600

2000

2400

2800

3200

3600N

3-N

cont

ent (

gＡminus

1D

W)

2 3 4 51

Time aer spraying (d)

(d)

Figure 2 Changes in the NRA andNO3-N content of burley tobacco underMo treatment during fast-growth period NRA nitrate reductaseactivity NRA in tobacco was determined at 6 h and on days 1ndash5 after spraying Error bars indicate standard error of means (119899 = 3)

growth led to significantly lower NO3-N content in KT204and TN86 Spraying Mo during the fast-growth period andsimultaneously spraying Mo and Gfo at the stage of maturityled to a significant decrease in NO3-N and NO2-N content inKT204 and TN86

35 Effects of Chemical Regulation on TSNAContents Auxinnaphthylacetic acid salicylic acid and malonic acid havebeen previously applied to decrease TSNA formation butthese may affect tobacco development and growth yield orquality [55 56] In this study we aimed to characterize achemical regulation strategy for decreasing TSNA precursors

so as to diminish TSNA formation in tobacco Yearly differ-ences in NO3-N NO2-N and TSNA contents in flue-curedtobacco were significant but TN and alkaloid levels werenot (Table 2) Regulatory treatments significantly affectedTN NO3-N alkaloid level and TSNA concentrations in flue-cured tobacco Varieties of burley tobacco were differentin TN NO3-N NO2-N alkaloid level and TSNA concen-trations and chemical regulation treatments significantlyaffected TN NO3-N and TSNA concentrations

As can be seen in Figure 5 spraying Mo during the fast-growth period and spraying Gfo at the stage of maturitydecreased TSNA concentrations in flue-cured tobacco but

Journal of Chemistry 7

CK

lowast

lowastlowast

lowastlowast

FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(a)

lowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

004

005

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(b)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

AVR

(gＧ

minus2Ｂminus1)

(c)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(d)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(e)

lowastlowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(f)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

40

AVR

(gＧ

minus2Ｂminus1)

(g)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(h)

Figure 3 Effects of Mo and Gfo treatments on NRA AVR GSA and SPRO in burley tobacco Error bars represent standard error (119899 = 3)NRA nitrate reductase activity AVR ammonia volatilization rate GSA glutamine synthetase activity SPRO total soluble protein content(andashd) KT204 (endashh) TN86 Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

8 Journal of Chemistry

Table 2 ANOVA comparison of the effects of regulatory treatments year and tobacco variety and their interactions on TN NO3-N NO2-Nalkaloid and TSNA concentrations in tobacco

Types Effect TN NO3-N NO2-N Alkaloid TSNAs DF

Flue-cured tobaccoYear (Y) 005ns 020lowast 001lowastlowast 160ns 004lowastlowast 1

Treatment (T) 901lowastlowast 1533lowastlowast 021ns 465lowast 719lowastlowast 3Year (Y) times Treatment (T) 854lowastlowast 5561lowastlowast 1371lowastlowast 573lowastlowast 3052lowastlowast 7

Burley tobaccoVariety (V) 097lowast 018lowast 1108lowastlowast 002lowastlowast 118lowast 1

Treatment (T) 359lowast 1934lowastlowast 097ns 028ns 1868lowastlowast 3Variety (V) times Treatment (T) 566lowastlowast 27557lowastlowast 2841lowastlowast 1314lowastlowast 27477lowastlowast 7

119865-values and significance levels are given (lowastlowast119901 lt 001 lowast119901 lt 005 and ns119901 ge 005)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(a)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowastlowastlowast

lowastlowast

0

500

1000

1500

2000

2500

3000

3500

N

3-N

cont

ent (

gＡminus

1D

W)

(b)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowast

0

1

2

3

4

N

2-N

cont

ent (

gＡminus

1D

W)

(c)M

-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1

2

3

4

Alk

aloi

d co

nten

t (

DW

)

(d)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

8

N

3-N

TN

()

(e)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(f)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

500

1000

1500

2000

2500

N

3-N

cont

ent (

gＡminus

1D

W)

(g)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowastlowast

00

05

10

15

20

N

2-N

cont

ent (

gＡminus

1D

W)

(h)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

2

4

6

Alk

aloi

d co

nten

t (

DW

)

(i)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

N

3-N

TN

()

(j)

Figure 4 Effects of treatments on total nitrogen content NO3-N content NO2-N content alkaloid content and NO3-NTN in burleytobacco NO3-NTN ratio of NO3-N and total nitrogen content (TN) Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicatesignificant difference at 001 or 005 respectively

the effect of spraying Mo during the fast-growth period wassignificantly different (119901 lt 005) Spraying Mo during thefast-growth period and spraying Mo and Gfo at maturityproduced the best results on TSNA concentrations among alltreatments in both 2014 and 2015 Spraying Mo during thefast-growth period could significantly reduce concentrations

of NNN NAB andNAT SprayingMo during the fast-growthperiod and spraying Gfo at the stage of maturity decreasedNNNNABNAT and total TSNA concentration in both 2014and 2015 respectively

TSNA accumulation in burley tobacco was much higherthan in flue-cured tobacco However effects of regulatory

Journal of Chemistry 9

2014

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

50

60

70

80

90

100

NN

N (n

gＡminus

1D

W)

(a)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

25

30

35

40

NAT

(ngＡminus

1D

W)

(b)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

20

25

30

35

40

NA

B (n

gＡminus

1D

W)

(c)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowastlowastlowast

lowastlowast

20

30

40

50

NN

K (n

gＡminus

1D

W)

(d)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast lowastlowast

lowastlowast

100

125

150

175

200

TSN

As (

ngＡminus

1D

W)

(e)2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

40

50

60

70

80

90

NN

N (n

gＡminus

1D

W)

(f)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

30

40

50

60

70

80

90

100

NAT

(ngＡminus

1D

W)

(g)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

10

15

20

25

30

NA

B (n

gＡminus

1D

W)

(h)

2015

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK20

25

30

35

40

45

50

55

60

NN

K (n

gＡminus

1D

W)

(i)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

80

120

160

200

240

TSN

As (

ngＡminus

1D

W)(j)

Figure 5 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in flue-cured tobacco Error bars indicatestandard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

treatment were more pronounced in burley tobacco Withinburley varieties the TSNA concentrations in KT204 werehigher than that in TN86 (Figures 6(a)ndash6(e)) Spraying ofModuring the fast-growth period led to significantly lowerNNNNAB and total TSNA concentrations in KT204 Sprayingof Gfo at maturity led to significant decreases in NNNNAT NNK and TSNA concentrations in TN86 The TSNA-regulating effects of the two treatments were optimized byspraying Mo during the fast-growth period and Gfo at thestage of maturity NNN NAT NAB NNK and total TSNAconcentration decreased in KT204 and TN86

36 Correlation Analysis Linear relationships betweenTSNAs alkaloids and NO3-N were significantly different(Figures 7(a)ndash7(c)) Total TSNA concentration in tobaccoincreased with increasing alkaloid and NO3-N contentespecially in burley tobacco The positive correlations

between TSNAs and their precursors were also reported byLewis et al [57] who suggested that NO3-N was a strongercontributing factor to higher TSNA levels than increasedalkaloid levels in burley tobacco

4 Conclusion

Nitrate was higher in burley tobacco than in flue-curedtobacco with both types showing peak nitrate content duringthe fast-growth period Under Mo treatment at the stage ofmaturity to avoid nitrate accumulation NRA LDM and DMin tobacco leaves increased SprayingMo in combinationwithGfo at the stage of maturity led to increased NRA and lowerGSA in tobacco which could help decrease nitrate and nitritecontent by increasing nitrogen loss via ammonia volatiliza-tion In summary spraying Mo during fast growth andspraying Mo with Gfo at the stage of maturity were effectivein reducing the formation of TSNAs

10 Journal of Chemistry

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

500

1000

1500

2000

NN

N (n

gＡminus

1D

W)

(a)

lowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

10

20

30

NA

B (n

gＡminus

1D

W)

(b)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

200

400

600

NAT

(ngＡminus

1D

W)

(c)

lowastlowastlowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(d)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1000

2000

3000

TSN

As (

ngＡminus

1D

W)

(e)

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

NN

N (n

gＡminus

1D

W)

(f)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

10

20

30

NA

B (n

gＡminus

1D

W)

(g)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

200

400

600

800N

AT (n

gＡminus

1D

W)

(h)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(i)

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

4000

TSN

As (

ngＡminus

1D

W)

(j)

Figure 6 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in burley tobacco varieties TN86 andKT204 Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

Burley tobacco

Flue-cured tobacco

R2 = 07220lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

3 4

Alkaloid content ( DW)

y = 15176x minus 35891

(a)

Burley tobacco

Flue-cured tobacco

R2 = 00557

0

2000

4000

TSN

As (

ngＡminus

1D

W)

321

N2-N content (g Ａminus1 DW)

y = 48834x + 17464

(b)

N3-N content (g Ａminus1 DW)3000200010000

Burley tobacco

Flue-cured tobacco

R2 = 07983lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

y = 121643x minus 28745602

(c)

Figure 7 Correlation analysis between TSNAs alkaloid NO2-N and NO3-N in tobacco Symbol lowastlowast indicates significant correlation at119901 lt 001

Journal of Chemistry 11

Gln

NR NiR

Glu Glu

Protein

Gln 2OG

NR NiRGS

TCA

TSNAs

Mo

NR Protein

NR GS

In fastgrowthperiod

At maturitystage

Leaf

Leaf

Nitratestorage

Nitrateaccumulation

Leaf

Nitratetransport

Glu Glu

Leaf

2OG

N（3

N（3

N（3

N3-N

N3-N

N3-N

N2-N

N2-N

Nx + lkaloid

MＩ + fo

Figure 8 Mechanisms for decreasing nitrate and TSNA concentrations in tobacco by spraying regulating chemicals Gfo glufosinate NRnitrate reductase NRA nitrate reductase activity NiR nitrite reductase GS glutamine synthetase GSA glutamine synthetase activity Glnglutamine Glu glutamate and OG oxaloacetate After sprayingMo on tobacco during the fast-growth period nitrate significantly decreasedwhile NRA and soluble protein content increasedThese decreased the amount of nitrate storage and promoted tobacco development duringthe fast-growth period After spraying Mo during the fast-growth stage and spraying Mo and Gfo at the stage of maturity NRA increasedand GSA decreased in tobacco which can significantly reduce nitrate accumulation and TSNA formation by nitrogen loss due to ammoniavolatilization

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank Editage (httpswwweditagecom) forEnglish language editing and publication support

References

[1] H R Burton N K Dye and L P Bush ldquoRelationship betweentobacco-specific nitrosamines and nitrite from different air-cured tobacco varietiesrdquo Journal of Agricultural and FoodChemistry vol 42 no 9 pp 2007ndash2011 1994

[2] B Siminszky L Gavilano S W Bowen and R E DeweyldquoConversion of nicotine to nornicotine in Nicotiana tabacumis mediated by CYP82E4 a cytochrome P450 monooxygenaserdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 41 pp 14919ndash14924 2005

[3] IARC ldquoSmokeless tobacco and some tobacco-specific N-nitrosamines monographs on the evaluation of carcinogenicrisks to humansrdquo National Publishing Group vol 89 Article ID1e592 2005

[4] H Shi R Wang and L P Bush ldquoThe relationships betweenTSNAs and their precursors in burley tobacco from differentregions and varieties JournalofFoodrdquoAgricultureEnvironmentvol 10 no 2 pp 1048ndash1052 2012

[5] H R Burton G H Childs Jr R A Andersen and P D Flem-ing ldquoChanges in chemical composition of Burley tobacco dur-ing senescence and curing 3 Tobacco-specific nitrosaminesrdquoJournal of Agricultural and Food Chemistry vol 37 no 2 pp426ndash430 1989

[6] H Shi R Wang L P Bush et al ldquoChanges in TSNA Contentsduring Tobacco Storage and the Effect of Temperature andNitrate Level on TSNA Formationrdquo Journal of Agricultural andFood Chemistry vol 61 pp 11588ndash11594 2013

[7] K Fytianos and P Zarogiannis ldquoNitrate and nitrite accumula-tion in fresh vegetables from Greecerdquo Bulletin of EnvironmentalContamination and Toxicology vol 62 no 2 pp 187ndash192 1999

[8] Y Y Wang P K Hsu and Y F Tsay ldquoUptake allocation andsignaling of nitraterdquo Trends in Plant Science vol 17 no 8 pp458ndash467 2012

[9] K S Reddy and R C Menary ldquoNitrate reductase and nitrateaccumulation in relation to nitrate toxicity in Boronia meg-astigmardquo Physiologia Plantarum vol 78 no 3 pp 430ndash4341990

[10] Y Han Q Liao Y Yu et al ldquoNitrate reutilization mechanismsin the tonoplast of two Brassica napus genotypes with differentnitrogen use efficiencyrdquoActa Physiologiae Plantarum vol 37 no2 2015

[11] H Kosaka K Imaizumi K Imai and I Tyuma ldquoStoichiometryof the reaction of oxyhemoglobin with nitriterdquo BBA - ProteinStructure vol 581 no 1 pp 184ndash188 1979

[12] P Santamaria ldquoNitrate in vegetables toxicity content intakeand EC regulationrdquo Journal of the Science of Food and Agricul-ture vol 86 no 1 pp 10ndash17 2006

[13] A T Diplock P J Aggett M Ashwell F Bornet E B FernandM B Roberfroid ldquoScientific concepts of functional foods in

4 Journal of Chemistry

Table 1 ANOVA results of the effects of chemical regulation year and tobacco variety and their interactions on LDMandDMbefore harvest

Year Treatment LDM (gplant) DM (gplant) Variety Treatment LDM (gplant) DM (gplant)

2014

CK 13070 plusmn 733b 24070 plusmn 791a

KT204

CK 12413 plusmn 326ab 24413 plusmn 442a

FG-Mo 14935 plusmn 349a 26035 plusmn 378a FG-Mo 13405 plusmn 352a 25605 plusmn 468a

M-Gfo 11433 plusmn 275c 22433 plusmn 289b M-Gfo 11849 plusmn 226b 23749 plusmn 441a

M-Mo + Gfo 13785 plusmn 278ab 24785 plusmn 335a M-Mo + Gfo 12817 plusmn 315ab 24817 plusmn 430a

2015

CK 14270 plusmn 584a 25570 plusmn 755a

TN86

CK 11341 plusmn 454ab 21541 plusmn 569ab

FG-Mo 15527 plusmn 499a 26860 plusmn 701a FG-Mo 12534 plusmn 292a 23301 plusmn 437a

M-Gfo 14102 plusmn 430a 25402 plusmn 603a M-Gfo 10413 plusmn 338b 20413 plusmn 453b

M-Mo + Gfo 15152 plusmn 720a 26452 plusmn 893a M-Mo + Gfo 11665 plusmn 328ab 22065 plusmn 558ab

Year (Y) 202lowastlowast 065lowastlowast Variety (V) 090lowast 194lowastlowast

Treatment (T) 471lowastlowast 344lowast Treatment (T) 528lowastlowast 199

Year (Y) times treatment (T) 671lowastlowast 513lowastlowast Variety (V) times treatment (T) 788lowastlowast 1438lowastlowast

Different letters within the same column indicate significant differences among treatments at 119901 lt 005 Symbols lowastlowast and lowast indicate significant difference at 001or 005 respectively

increased over the period of development and presented atrend of ldquorise-fallrdquo prior to harvest (Figure 1) Nitrate contentwas at its highest during the fast-growth period Nitrate isdifficult to recycle once stored in cells [48] Hence avoidingnitrate accumulation during the fast-growth stage may beeffective in reducing nitrate accumulation in cured tobacco

In general the amount of nitrogen fertilizers used onburley tobacco was almost 3ndash5 times higher than that used onflue-cured tobacco but the yield was not significantly differ-ent between them [49] NRA and NO3-N contents betweenburley tobacco and flue-cured tobacco were significantlydifferent with NRANA in flue-cured tobacco significantlyhigher than in burley tobacco During tobacco developmentthe NO3-N content in burley tobacco was higher than that influe-cured tobacco in both field and pot experiments NRAwas readily affected by nitrogen application with nitrogenapplication on burley tobacco 4-fold greater than that in flue-cured tobacco production NRANA in flue-cured tobaccowas higher than in burley tobacco in both field and pot exper-iments In addition weak nitrogen assimilation of burleytobacco may be an important cause of nitrate accumulation[50]

32 Effects of Chemical Regulation on Leaf Biological Yield(LDM) and Above-Ground Dry Matter Weight (DM) LDMand DM were used to evaluate whether plants were growingwell and to predict yield in tobacco cultivation [36] It hasbeen reported thatDM yield and product quality all decreaseunder a Mo-deficient condition [51] In this work LDM andDM increased with Mo being sprayed during the fast-growthperiod which has been shown to dilute nitrate concentration[50] The main effects of chemical treatment and year weresignificantly observed for LDM andDMover the two years ofobservation (119901 lt 005) (Table 1) Variation between tobaccovarieties also significantly affected LDM and DM LDM andDMin tobacco increased underMo treatment during the fast-growth period Meanwhile LDM and DM showed a decreasewith spraying of Gfo at the maturity stage

33 Effects of Chemical Regulation on NRA GSA AVR SPROandNO3-NContent NRA andNO3-N content in both TN86and KT204 exhibited increasing trends (Figure 2) whichwere closely related to the maximum uptake of nutrientsduring the rapid growth stage [36] Additionally enhancingnitrogen assimilation ability and decreasing nitrate storagewere key in reducing nitrate accumulation in tobacco duringthis period Under the Mo treatment during the fast-growthperiod NRA in TN86 and KT204 increased by 157ndash1181and 172ndash1058 respectively but NO3-N content in TN86and KT204 decreased correspondingly by 1016ndash5808 and1004ndash4887 respectively (119901 lt 001)

Composition of tobacco at the stage of maturity is signif-icantly indicative of the components of cured tobacco andimproving chemical composition during this stage is useful inenhancing tobacco quality [52] NR and GS are key enzymesin the process of nitrogen reduction and assimilation inplants and GS plays an important role in the first step ofNH4+ assimilation [53] NRA AVR GSA and SPRO in

burley tobacco were significantly affected by spraying Gfo atthe maturity stage (Figures 3(a)ndash3(h)) Gfo application caninhibit GSA and cause ammonia emissions of almost 10 ofcanopy nitrogen content [26] Compared with CK the GSAand SPRO of Gfo-sprayed tobacco significantly decreasedandAVR significantly increasedHence sprayingMo andGfoat maturity was effective in decreasing nitrate accumulationand promoting nitrogen loss in tobacco (Figure 8)

34 Effects of Chemical Regulation on TSNA PrecursorsNO3-N NO2-N and alkaloids are precursors of TSNAsand decreasing precursors is effective in reducing TSNAformation in tobacco Sufficient NO3-N content can greatlypromote TSNA formation during tobacco storage andreducing NO3-N accumulation is key in decreasing TSNAformation [54] As shown above treatment with Mo andGfo significantly decreased TN NO3-N NO2-N and NO3-NTN but did not affect alkaloid levels in burley tobacco(Figures 4(a)ndash4(j)) Spraying Mo during periods of fast

Journal of Chemistry 5

Field experiment

807570656055504540353025

Days aer transplantation (d)

0

10

20

30

NRA

NA

40

50

60

70

HDY87

KT204

TN86

(a)

Pot experiment

656055504540353025201510

Days aer transplantation (d)

0

10

20NRA

NA

30

40

50

60

HDY87

KT204

TN86

(b)

Field experiment

807570656055504540353025

Days aer transplantation (d)

50

100

150

200

250

300

NRA

(g

mＡminus

1Ｂminus1

FW)

HDY87

KT204

TN86

(c)

Pot experiment

656055504540353025201510

Days aer transplantation (d)

50

75

100

125

150

175

200N

RA (

g mＡminus

1Ｂminus1

FW)

HDY87

KT204

TN86

(d)

Field experiment

N

3-N

cont

ent (

gＡminus

1D

W) 5000

4000

3000

2000

1000

075604530 Before

Days aer transplantation (d)

HDY87

KT204

TN86

harvest

(e)

Pot experiment

60453015

Days aer transplantation (d)

Beforeharvest

0

1000

2000

3000

4000

5000

N

3-N

cont

ent (

gＡminus

1D

W)

HDY87

KT204

TN86

(f)

Figure 1 Difference between burley tobacco and flue-cured tobacco inNRANRANA andNO3-N content of leaves Burley tobacco varietieswere KT204 and TN86 and flue-cured tobacco varieties were HD and Y87 NA nitrogen application (HD and Y87 45 kg haminus2 KT204 andTN86 180 kg haminus2) NRA nitrate reductase activity Error bars indicate standard error of the means (119899 = 3)

6 Journal of Chemistry

TN86

CKFG-Mo

175

200

225

250

275

300

NRA

(g

mＡminus

1Ｂminus1

FW)

1 d 2 d 3 d 4 d 5 d6 hTime aer spraying

(a)

KT204

CKFG-Mo

200

225

250

275

300

NRA

(g

mＡminus

1Ｂminus1

FW)

1 d 2 d 3 d 4 d 5 d6 hTime aer spraying

(b)

TN86

CKFG-Mo

1600

2400

3200

4000

4800

N

3-N

cont

ent (

gＡminus

1D

W)

2 3 4 51

Time aer spraying (d)

(c)

KT204

CKFG-Mo

1600

2000

2400

2800

3200

3600N

3-N

cont

ent (

gＡminus

1D

W)

2 3 4 51

Time aer spraying (d)

(d)

Figure 2 Changes in the NRA andNO3-N content of burley tobacco underMo treatment during fast-growth period NRA nitrate reductaseactivity NRA in tobacco was determined at 6 h and on days 1ndash5 after spraying Error bars indicate standard error of means (119899 = 3)

growth led to significantly lower NO3-N content in KT204and TN86 Spraying Mo during the fast-growth period andsimultaneously spraying Mo and Gfo at the stage of maturityled to a significant decrease in NO3-N and NO2-N content inKT204 and TN86

35 Effects of Chemical Regulation on TSNAContents Auxinnaphthylacetic acid salicylic acid and malonic acid havebeen previously applied to decrease TSNA formation butthese may affect tobacco development and growth yield orquality [55 56] In this study we aimed to characterize achemical regulation strategy for decreasing TSNA precursors

so as to diminish TSNA formation in tobacco Yearly differ-ences in NO3-N NO2-N and TSNA contents in flue-curedtobacco were significant but TN and alkaloid levels werenot (Table 2) Regulatory treatments significantly affectedTN NO3-N alkaloid level and TSNA concentrations in flue-cured tobacco Varieties of burley tobacco were differentin TN NO3-N NO2-N alkaloid level and TSNA concen-trations and chemical regulation treatments significantlyaffected TN NO3-N and TSNA concentrations

As can be seen in Figure 5 spraying Mo during the fast-growth period and spraying Gfo at the stage of maturitydecreased TSNA concentrations in flue-cured tobacco but

Journal of Chemistry 7

CK

lowast

lowastlowast

lowastlowast

FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(a)

lowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

004

005

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(b)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

AVR

(gＧ

minus2Ｂminus1)

(c)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(d)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(e)

lowastlowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(f)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

40

AVR

(gＧ

minus2Ｂminus1)

(g)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(h)

Figure 3 Effects of Mo and Gfo treatments on NRA AVR GSA and SPRO in burley tobacco Error bars represent standard error (119899 = 3)NRA nitrate reductase activity AVR ammonia volatilization rate GSA glutamine synthetase activity SPRO total soluble protein content(andashd) KT204 (endashh) TN86 Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

8 Journal of Chemistry

Table 2 ANOVA comparison of the effects of regulatory treatments year and tobacco variety and their interactions on TN NO3-N NO2-Nalkaloid and TSNA concentrations in tobacco

Types Effect TN NO3-N NO2-N Alkaloid TSNAs DF

Flue-cured tobaccoYear (Y) 005ns 020lowast 001lowastlowast 160ns 004lowastlowast 1

Treatment (T) 901lowastlowast 1533lowastlowast 021ns 465lowast 719lowastlowast 3Year (Y) times Treatment (T) 854lowastlowast 5561lowastlowast 1371lowastlowast 573lowastlowast 3052lowastlowast 7

Burley tobaccoVariety (V) 097lowast 018lowast 1108lowastlowast 002lowastlowast 118lowast 1

Treatment (T) 359lowast 1934lowastlowast 097ns 028ns 1868lowastlowast 3Variety (V) times Treatment (T) 566lowastlowast 27557lowastlowast 2841lowastlowast 1314lowastlowast 27477lowastlowast 7

119865-values and significance levels are given (lowastlowast119901 lt 001 lowast119901 lt 005 and ns119901 ge 005)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(a)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowastlowastlowast

lowastlowast

0

500

1000

1500

2000

2500

3000

3500

N

3-N

cont

ent (

gＡminus

1D

W)

(b)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowast

0

1

2

3

4

N

2-N

cont

ent (

gＡminus

1D

W)

(c)M

-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1

2

3

4

Alk

aloi

d co

nten

t (

DW

)

(d)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

8

N

3-N

TN

()

(e)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(f)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

500

1000

1500

2000

2500

N

3-N

cont

ent (

gＡminus

1D

W)

(g)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowastlowast

00

05

10

15

20

N

2-N

cont

ent (

gＡminus

1D

W)

(h)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

2

4

6

Alk

aloi

d co

nten

t (

DW

)

(i)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

N

3-N

TN

()

(j)

Figure 4 Effects of treatments on total nitrogen content NO3-N content NO2-N content alkaloid content and NO3-NTN in burleytobacco NO3-NTN ratio of NO3-N and total nitrogen content (TN) Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicatesignificant difference at 001 or 005 respectively

the effect of spraying Mo during the fast-growth period wassignificantly different (119901 lt 005) Spraying Mo during thefast-growth period and spraying Mo and Gfo at maturityproduced the best results on TSNA concentrations among alltreatments in both 2014 and 2015 Spraying Mo during thefast-growth period could significantly reduce concentrations

of NNN NAB andNAT SprayingMo during the fast-growthperiod and spraying Gfo at the stage of maturity decreasedNNNNABNAT and total TSNA concentration in both 2014and 2015 respectively

TSNA accumulation in burley tobacco was much higherthan in flue-cured tobacco However effects of regulatory

Journal of Chemistry 9

2014

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

50

60

70

80

90

100

NN

N (n

gＡminus

1D

W)

(a)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

25

30

35

40

NAT

(ngＡminus

1D

W)

(b)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

20

25

30

35

40

NA

B (n

gＡminus

1D

W)

(c)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowastlowastlowast

lowastlowast

20

30

40

50

NN

K (n

gＡminus

1D

W)

(d)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast lowastlowast

lowastlowast

100

125

150

175

200

TSN

As (

ngＡminus

1D

W)

(e)2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

40

50

60

70

80

90

NN

N (n

gＡminus

1D

W)

(f)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

30

40

50

60

70

80

90

100

NAT

(ngＡminus

1D

W)

(g)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

10

15

20

25

30

NA

B (n

gＡminus

1D

W)

(h)

2015

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK20

25

30

35

40

45

50

55

60

NN

K (n

gＡminus

1D

W)

(i)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

80

120

160

200

240

TSN

As (

ngＡminus

1D

W)(j)

Figure 5 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in flue-cured tobacco Error bars indicatestandard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

treatment were more pronounced in burley tobacco Withinburley varieties the TSNA concentrations in KT204 werehigher than that in TN86 (Figures 6(a)ndash6(e)) Spraying ofModuring the fast-growth period led to significantly lowerNNNNAB and total TSNA concentrations in KT204 Sprayingof Gfo at maturity led to significant decreases in NNNNAT NNK and TSNA concentrations in TN86 The TSNA-regulating effects of the two treatments were optimized byspraying Mo during the fast-growth period and Gfo at thestage of maturity NNN NAT NAB NNK and total TSNAconcentration decreased in KT204 and TN86

36 Correlation Analysis Linear relationships betweenTSNAs alkaloids and NO3-N were significantly different(Figures 7(a)ndash7(c)) Total TSNA concentration in tobaccoincreased with increasing alkaloid and NO3-N contentespecially in burley tobacco The positive correlations

between TSNAs and their precursors were also reported byLewis et al [57] who suggested that NO3-N was a strongercontributing factor to higher TSNA levels than increasedalkaloid levels in burley tobacco

4 Conclusion

Nitrate was higher in burley tobacco than in flue-curedtobacco with both types showing peak nitrate content duringthe fast-growth period Under Mo treatment at the stage ofmaturity to avoid nitrate accumulation NRA LDM and DMin tobacco leaves increased SprayingMo in combinationwithGfo at the stage of maturity led to increased NRA and lowerGSA in tobacco which could help decrease nitrate and nitritecontent by increasing nitrogen loss via ammonia volatiliza-tion In summary spraying Mo during fast growth andspraying Mo with Gfo at the stage of maturity were effectivein reducing the formation of TSNAs

10 Journal of Chemistry

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

500

1000

1500

2000

NN

N (n

gＡminus

1D

W)

(a)

lowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

10

20

30

NA

B (n

gＡminus

1D

W)

(b)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

200

400

600

NAT

(ngＡminus

1D

W)

(c)

lowastlowastlowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(d)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1000

2000

3000

TSN

As (

ngＡminus

1D

W)

(e)

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

NN

N (n

gＡminus

1D

W)

(f)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

10

20

30

NA

B (n

gＡminus

1D

W)

(g)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

200

400

600

800N

AT (n

gＡminus

1D

W)

(h)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(i)

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

4000

TSN

As (

ngＡminus

1D

W)

(j)

Figure 6 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in burley tobacco varieties TN86 andKT204 Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

Burley tobacco

Flue-cured tobacco

R2 = 07220lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

3 4

Alkaloid content ( DW)

y = 15176x minus 35891

(a)

Burley tobacco

Flue-cured tobacco

R2 = 00557

0

2000

4000

TSN

As (

ngＡminus

1D

W)

321

N2-N content (g Ａminus1 DW)

y = 48834x + 17464

(b)

N3-N content (g Ａminus1 DW)3000200010000

Burley tobacco

Flue-cured tobacco

R2 = 07983lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

y = 121643x minus 28745602

(c)

Figure 7 Correlation analysis between TSNAs alkaloid NO2-N and NO3-N in tobacco Symbol lowastlowast indicates significant correlation at119901 lt 001

Journal of Chemistry 11

Gln

NR NiR

Glu Glu

Protein

Gln 2OG

NR NiRGS

TCA

TSNAs

Mo

NR Protein

NR GS

In fastgrowthperiod

At maturitystage

Leaf

Leaf

Nitratestorage

Nitrateaccumulation

Leaf

Nitratetransport

Glu Glu

Leaf

2OG

N（3

N（3

N（3

N3-N

N3-N

N3-N

N2-N

N2-N

Nx + lkaloid

MＩ + fo

Figure 8 Mechanisms for decreasing nitrate and TSNA concentrations in tobacco by spraying regulating chemicals Gfo glufosinate NRnitrate reductase NRA nitrate reductase activity NiR nitrite reductase GS glutamine synthetase GSA glutamine synthetase activity Glnglutamine Glu glutamate and OG oxaloacetate After sprayingMo on tobacco during the fast-growth period nitrate significantly decreasedwhile NRA and soluble protein content increasedThese decreased the amount of nitrate storage and promoted tobacco development duringthe fast-growth period After spraying Mo during the fast-growth stage and spraying Mo and Gfo at the stage of maturity NRA increasedand GSA decreased in tobacco which can significantly reduce nitrate accumulation and TSNA formation by nitrogen loss due to ammoniavolatilization

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank Editage (httpswwweditagecom) forEnglish language editing and publication support

References

[1] H R Burton N K Dye and L P Bush ldquoRelationship betweentobacco-specific nitrosamines and nitrite from different air-cured tobacco varietiesrdquo Journal of Agricultural and FoodChemistry vol 42 no 9 pp 2007ndash2011 1994

[2] B Siminszky L Gavilano S W Bowen and R E DeweyldquoConversion of nicotine to nornicotine in Nicotiana tabacumis mediated by CYP82E4 a cytochrome P450 monooxygenaserdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 41 pp 14919ndash14924 2005

[3] IARC ldquoSmokeless tobacco and some tobacco-specific N-nitrosamines monographs on the evaluation of carcinogenicrisks to humansrdquo National Publishing Group vol 89 Article ID1e592 2005

[4] H Shi R Wang and L P Bush ldquoThe relationships betweenTSNAs and their precursors in burley tobacco from differentregions and varieties JournalofFoodrdquoAgricultureEnvironmentvol 10 no 2 pp 1048ndash1052 2012

[5] H R Burton G H Childs Jr R A Andersen and P D Flem-ing ldquoChanges in chemical composition of Burley tobacco dur-ing senescence and curing 3 Tobacco-specific nitrosaminesrdquoJournal of Agricultural and Food Chemistry vol 37 no 2 pp426ndash430 1989

[6] H Shi R Wang L P Bush et al ldquoChanges in TSNA Contentsduring Tobacco Storage and the Effect of Temperature andNitrate Level on TSNA Formationrdquo Journal of Agricultural andFood Chemistry vol 61 pp 11588ndash11594 2013

[7] K Fytianos and P Zarogiannis ldquoNitrate and nitrite accumula-tion in fresh vegetables from Greecerdquo Bulletin of EnvironmentalContamination and Toxicology vol 62 no 2 pp 187ndash192 1999

[8] Y Y Wang P K Hsu and Y F Tsay ldquoUptake allocation andsignaling of nitraterdquo Trends in Plant Science vol 17 no 8 pp458ndash467 2012

[9] K S Reddy and R C Menary ldquoNitrate reductase and nitrateaccumulation in relation to nitrate toxicity in Boronia meg-astigmardquo Physiologia Plantarum vol 78 no 3 pp 430ndash4341990

[10] Y Han Q Liao Y Yu et al ldquoNitrate reutilization mechanismsin the tonoplast of two Brassica napus genotypes with differentnitrogen use efficiencyrdquoActa Physiologiae Plantarum vol 37 no2 2015

[11] H Kosaka K Imaizumi K Imai and I Tyuma ldquoStoichiometryof the reaction of oxyhemoglobin with nitriterdquo BBA - ProteinStructure vol 581 no 1 pp 184ndash188 1979

[12] P Santamaria ldquoNitrate in vegetables toxicity content intakeand EC regulationrdquo Journal of the Science of Food and Agricul-ture vol 86 no 1 pp 10ndash17 2006

[13] A T Diplock P J Aggett M Ashwell F Bornet E B FernandM B Roberfroid ldquoScientific concepts of functional foods in

Journal of Chemistry 5

Field experiment

807570656055504540353025

Days aer transplantation (d)

0

10

20

30

NRA

NA

40

50

60

70

HDY87

KT204

TN86

(a)

Pot experiment

656055504540353025201510

Days aer transplantation (d)

0

10

20NRA

NA

30

40

50

60

HDY87

KT204

TN86

(b)

Field experiment

807570656055504540353025

Days aer transplantation (d)

50

100

150

200

250

300

NRA

(g

mＡminus

1Ｂminus1

FW)

HDY87

KT204

TN86

(c)

Pot experiment

656055504540353025201510

Days aer transplantation (d)

50

75

100

125

150

175

200N

RA (

g mＡminus

1Ｂminus1

FW)

HDY87

KT204

TN86

(d)

Field experiment

N

3-N

cont

ent (

gＡminus

1D

W) 5000

4000

3000

2000

1000

075604530 Before

Days aer transplantation (d)

HDY87

KT204

TN86

harvest

(e)

Pot experiment

60453015

Days aer transplantation (d)

Beforeharvest

0

1000

2000

3000

4000

5000

N

3-N

cont

ent (

gＡminus

1D

W)

HDY87

KT204

TN86

(f)

Figure 1 Difference between burley tobacco and flue-cured tobacco inNRANRANA andNO3-N content of leaves Burley tobacco varietieswere KT204 and TN86 and flue-cured tobacco varieties were HD and Y87 NA nitrogen application (HD and Y87 45 kg haminus2 KT204 andTN86 180 kg haminus2) NRA nitrate reductase activity Error bars indicate standard error of the means (119899 = 3)

6 Journal of Chemistry

TN86

CKFG-Mo

175

200

225

250

275

300

NRA

(g

mＡminus

1Ｂminus1

FW)

1 d 2 d 3 d 4 d 5 d6 hTime aer spraying

(a)

KT204

CKFG-Mo

200

225

250

275

300

NRA

(g

mＡminus

1Ｂminus1

FW)

1 d 2 d 3 d 4 d 5 d6 hTime aer spraying

(b)

TN86

CKFG-Mo

1600

2400

3200

4000

4800

N

3-N

cont

ent (

gＡminus

1D

W)

2 3 4 51

Time aer spraying (d)

(c)

KT204

CKFG-Mo

1600

2000

2400

2800

3200

3600N

3-N

cont

ent (

gＡminus

1D

W)

2 3 4 51

Time aer spraying (d)

(d)

Figure 2 Changes in the NRA andNO3-N content of burley tobacco underMo treatment during fast-growth period NRA nitrate reductaseactivity NRA in tobacco was determined at 6 h and on days 1ndash5 after spraying Error bars indicate standard error of means (119899 = 3)

growth led to significantly lower NO3-N content in KT204and TN86 Spraying Mo during the fast-growth period andsimultaneously spraying Mo and Gfo at the stage of maturityled to a significant decrease in NO3-N and NO2-N content inKT204 and TN86

35 Effects of Chemical Regulation on TSNAContents Auxinnaphthylacetic acid salicylic acid and malonic acid havebeen previously applied to decrease TSNA formation butthese may affect tobacco development and growth yield orquality [55 56] In this study we aimed to characterize achemical regulation strategy for decreasing TSNA precursors

so as to diminish TSNA formation in tobacco Yearly differ-ences in NO3-N NO2-N and TSNA contents in flue-curedtobacco were significant but TN and alkaloid levels werenot (Table 2) Regulatory treatments significantly affectedTN NO3-N alkaloid level and TSNA concentrations in flue-cured tobacco Varieties of burley tobacco were differentin TN NO3-N NO2-N alkaloid level and TSNA concen-trations and chemical regulation treatments significantlyaffected TN NO3-N and TSNA concentrations

As can be seen in Figure 5 spraying Mo during the fast-growth period and spraying Gfo at the stage of maturitydecreased TSNA concentrations in flue-cured tobacco but

Journal of Chemistry 7

CK

lowast

lowastlowast

lowastlowast

FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(a)

lowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

004

005

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(b)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

AVR

(gＧ

minus2Ｂminus1)

(c)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(d)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(e)

lowastlowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(f)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

40

AVR

(gＧ

minus2Ｂminus1)

(g)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(h)

Figure 3 Effects of Mo and Gfo treatments on NRA AVR GSA and SPRO in burley tobacco Error bars represent standard error (119899 = 3)NRA nitrate reductase activity AVR ammonia volatilization rate GSA glutamine synthetase activity SPRO total soluble protein content(andashd) KT204 (endashh) TN86 Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

8 Journal of Chemistry

Table 2 ANOVA comparison of the effects of regulatory treatments year and tobacco variety and their interactions on TN NO3-N NO2-Nalkaloid and TSNA concentrations in tobacco

Types Effect TN NO3-N NO2-N Alkaloid TSNAs DF

Flue-cured tobaccoYear (Y) 005ns 020lowast 001lowastlowast 160ns 004lowastlowast 1

Treatment (T) 901lowastlowast 1533lowastlowast 021ns 465lowast 719lowastlowast 3Year (Y) times Treatment (T) 854lowastlowast 5561lowastlowast 1371lowastlowast 573lowastlowast 3052lowastlowast 7

Burley tobaccoVariety (V) 097lowast 018lowast 1108lowastlowast 002lowastlowast 118lowast 1

Treatment (T) 359lowast 1934lowastlowast 097ns 028ns 1868lowastlowast 3Variety (V) times Treatment (T) 566lowastlowast 27557lowastlowast 2841lowastlowast 1314lowastlowast 27477lowastlowast 7

119865-values and significance levels are given (lowastlowast119901 lt 001 lowast119901 lt 005 and ns119901 ge 005)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(a)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowastlowastlowast

lowastlowast

0

500

1000

1500

2000

2500

3000

3500

N

3-N

cont

ent (

gＡminus

1D

W)

(b)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowast

0

1

2

3

4

N

2-N

cont

ent (

gＡminus

1D

W)

(c)M

-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1

2

3

4

Alk

aloi

d co

nten

t (

DW

)

(d)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

8

N

3-N

TN

()

(e)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(f)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

500

1000

1500

2000

2500

N

3-N

cont

ent (

gＡminus

1D

W)

(g)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowastlowast

00

05

10

15

20

N

2-N

cont

ent (

gＡminus

1D

W)

(h)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

2

4

6

Alk

aloi

d co

nten

t (

DW

)

(i)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

N

3-N

TN

()

(j)

Figure 4 Effects of treatments on total nitrogen content NO3-N content NO2-N content alkaloid content and NO3-NTN in burleytobacco NO3-NTN ratio of NO3-N and total nitrogen content (TN) Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicatesignificant difference at 001 or 005 respectively

the effect of spraying Mo during the fast-growth period wassignificantly different (119901 lt 005) Spraying Mo during thefast-growth period and spraying Mo and Gfo at maturityproduced the best results on TSNA concentrations among alltreatments in both 2014 and 2015 Spraying Mo during thefast-growth period could significantly reduce concentrations

of NNN NAB andNAT SprayingMo during the fast-growthperiod and spraying Gfo at the stage of maturity decreasedNNNNABNAT and total TSNA concentration in both 2014and 2015 respectively

TSNA accumulation in burley tobacco was much higherthan in flue-cured tobacco However effects of regulatory

Journal of Chemistry 9

2014

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

50

60

70

80

90

100

NN

N (n

gＡminus

1D

W)

(a)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

25

30

35

40

NAT

(ngＡminus

1D

W)

(b)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

20

25

30

35

40

NA

B (n

gＡminus

1D

W)

(c)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowastlowastlowast

lowastlowast

20

30

40

50

NN

K (n

gＡminus

1D

W)

(d)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast lowastlowast

lowastlowast

100

125

150

175

200

TSN

As (

ngＡminus

1D

W)

(e)2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

40

50

60

70

80

90

NN

N (n

gＡminus

1D

W)

(f)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

30

40

50

60

70

80

90

100

NAT

(ngＡminus

1D

W)

(g)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

10

15

20

25

30

NA

B (n

gＡminus

1D

W)

(h)

2015

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK20

25

30

35

40

45

50

55

60

NN

K (n

gＡminus

1D

W)

(i)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

80

120

160

200

240

TSN

As (

ngＡminus

1D

W)(j)

Figure 5 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in flue-cured tobacco Error bars indicatestandard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

treatment were more pronounced in burley tobacco Withinburley varieties the TSNA concentrations in KT204 werehigher than that in TN86 (Figures 6(a)ndash6(e)) Spraying ofModuring the fast-growth period led to significantly lowerNNNNAB and total TSNA concentrations in KT204 Sprayingof Gfo at maturity led to significant decreases in NNNNAT NNK and TSNA concentrations in TN86 The TSNA-regulating effects of the two treatments were optimized byspraying Mo during the fast-growth period and Gfo at thestage of maturity NNN NAT NAB NNK and total TSNAconcentration decreased in KT204 and TN86

36 Correlation Analysis Linear relationships betweenTSNAs alkaloids and NO3-N were significantly different(Figures 7(a)ndash7(c)) Total TSNA concentration in tobaccoincreased with increasing alkaloid and NO3-N contentespecially in burley tobacco The positive correlations

between TSNAs and their precursors were also reported byLewis et al [57] who suggested that NO3-N was a strongercontributing factor to higher TSNA levels than increasedalkaloid levels in burley tobacco

4 Conclusion

Nitrate was higher in burley tobacco than in flue-curedtobacco with both types showing peak nitrate content duringthe fast-growth period Under Mo treatment at the stage ofmaturity to avoid nitrate accumulation NRA LDM and DMin tobacco leaves increased SprayingMo in combinationwithGfo at the stage of maturity led to increased NRA and lowerGSA in tobacco which could help decrease nitrate and nitritecontent by increasing nitrogen loss via ammonia volatiliza-tion In summary spraying Mo during fast growth andspraying Mo with Gfo at the stage of maturity were effectivein reducing the formation of TSNAs

10 Journal of Chemistry

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

500

1000

1500

2000

NN

N (n

gＡminus

1D

W)

(a)

lowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

10

20

30

NA

B (n

gＡminus

1D

W)

(b)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

200

400

600

NAT

(ngＡminus

1D

W)

(c)

lowastlowastlowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(d)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1000

2000

3000

TSN

As (

ngＡminus

1D

W)

(e)

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

NN

N (n

gＡminus

1D

W)

(f)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

10

20

30

NA

B (n

gＡminus

1D

W)

(g)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

200

400

600

800N

AT (n

gＡminus

1D

W)

(h)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(i)

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

4000

TSN

As (

ngＡminus

1D

W)

(j)

Figure 6 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in burley tobacco varieties TN86 andKT204 Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

Burley tobacco

Flue-cured tobacco

R2 = 07220lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

3 4

Alkaloid content ( DW)

y = 15176x minus 35891

(a)

Burley tobacco

Flue-cured tobacco

R2 = 00557

0

2000

4000

TSN

As (

ngＡminus

1D

W)

321

N2-N content (g Ａminus1 DW)

y = 48834x + 17464

(b)

N3-N content (g Ａminus1 DW)3000200010000

Burley tobacco

Flue-cured tobacco

R2 = 07983lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

y = 121643x minus 28745602

(c)

Figure 7 Correlation analysis between TSNAs alkaloid NO2-N and NO3-N in tobacco Symbol lowastlowast indicates significant correlation at119901 lt 001

Journal of Chemistry 11

Gln

NR NiR

Glu Glu

Protein

Gln 2OG

NR NiRGS

TCA

TSNAs

Mo

NR Protein

NR GS

In fastgrowthperiod

At maturitystage

Leaf

Leaf

Nitratestorage

Nitrateaccumulation

Leaf

Nitratetransport

Glu Glu

Leaf

2OG

N（3

N（3

N（3

N3-N

N3-N

N3-N

N2-N

N2-N

Nx + lkaloid

MＩ + fo

Figure 8 Mechanisms for decreasing nitrate and TSNA concentrations in tobacco by spraying regulating chemicals Gfo glufosinate NRnitrate reductase NRA nitrate reductase activity NiR nitrite reductase GS glutamine synthetase GSA glutamine synthetase activity Glnglutamine Glu glutamate and OG oxaloacetate After sprayingMo on tobacco during the fast-growth period nitrate significantly decreasedwhile NRA and soluble protein content increasedThese decreased the amount of nitrate storage and promoted tobacco development duringthe fast-growth period After spraying Mo during the fast-growth stage and spraying Mo and Gfo at the stage of maturity NRA increasedand GSA decreased in tobacco which can significantly reduce nitrate accumulation and TSNA formation by nitrogen loss due to ammoniavolatilization

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank Editage (httpswwweditagecom) forEnglish language editing and publication support

References

[1] H R Burton N K Dye and L P Bush ldquoRelationship betweentobacco-specific nitrosamines and nitrite from different air-cured tobacco varietiesrdquo Journal of Agricultural and FoodChemistry vol 42 no 9 pp 2007ndash2011 1994

[2] B Siminszky L Gavilano S W Bowen and R E DeweyldquoConversion of nicotine to nornicotine in Nicotiana tabacumis mediated by CYP82E4 a cytochrome P450 monooxygenaserdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 41 pp 14919ndash14924 2005

[3] IARC ldquoSmokeless tobacco and some tobacco-specific N-nitrosamines monographs on the evaluation of carcinogenicrisks to humansrdquo National Publishing Group vol 89 Article ID1e592 2005

[4] H Shi R Wang and L P Bush ldquoThe relationships betweenTSNAs and their precursors in burley tobacco from differentregions and varieties JournalofFoodrdquoAgricultureEnvironmentvol 10 no 2 pp 1048ndash1052 2012

[5] H R Burton G H Childs Jr R A Andersen and P D Flem-ing ldquoChanges in chemical composition of Burley tobacco dur-ing senescence and curing 3 Tobacco-specific nitrosaminesrdquoJournal of Agricultural and Food Chemistry vol 37 no 2 pp426ndash430 1989

[6] H Shi R Wang L P Bush et al ldquoChanges in TSNA Contentsduring Tobacco Storage and the Effect of Temperature andNitrate Level on TSNA Formationrdquo Journal of Agricultural andFood Chemistry vol 61 pp 11588ndash11594 2013

[7] K Fytianos and P Zarogiannis ldquoNitrate and nitrite accumula-tion in fresh vegetables from Greecerdquo Bulletin of EnvironmentalContamination and Toxicology vol 62 no 2 pp 187ndash192 1999

[8] Y Y Wang P K Hsu and Y F Tsay ldquoUptake allocation andsignaling of nitraterdquo Trends in Plant Science vol 17 no 8 pp458ndash467 2012

[9] K S Reddy and R C Menary ldquoNitrate reductase and nitrateaccumulation in relation to nitrate toxicity in Boronia meg-astigmardquo Physiologia Plantarum vol 78 no 3 pp 430ndash4341990

[10] Y Han Q Liao Y Yu et al ldquoNitrate reutilization mechanismsin the tonoplast of two Brassica napus genotypes with differentnitrogen use efficiencyrdquoActa Physiologiae Plantarum vol 37 no2 2015

[11] H Kosaka K Imaizumi K Imai and I Tyuma ldquoStoichiometryof the reaction of oxyhemoglobin with nitriterdquo BBA - ProteinStructure vol 581 no 1 pp 184ndash188 1979

[12] P Santamaria ldquoNitrate in vegetables toxicity content intakeand EC regulationrdquo Journal of the Science of Food and Agricul-ture vol 86 no 1 pp 10ndash17 2006

[13] A T Diplock P J Aggett M Ashwell F Bornet E B FernandM B Roberfroid ldquoScientific concepts of functional foods in

6 Journal of Chemistry

TN86

CKFG-Mo

175

200

225

250

275

300

NRA

(g

mＡminus

1Ｂminus1

FW)

1 d 2 d 3 d 4 d 5 d6 hTime aer spraying

(a)

KT204

CKFG-Mo

200

225

250

275

300

NRA

(g

mＡminus

1Ｂminus1

FW)

1 d 2 d 3 d 4 d 5 d6 hTime aer spraying

(b)

TN86

CKFG-Mo

1600

2400

3200

4000

4800

N

3-N

cont

ent (

gＡminus

1D

W)

2 3 4 51

Time aer spraying (d)

(c)

KT204

CKFG-Mo

1600

2000

2400

2800

3200

3600N

3-N

cont

ent (

gＡminus

1D

W)

2 3 4 51

Time aer spraying (d)

(d)

Figure 2 Changes in the NRA andNO3-N content of burley tobacco underMo treatment during fast-growth period NRA nitrate reductaseactivity NRA in tobacco was determined at 6 h and on days 1ndash5 after spraying Error bars indicate standard error of means (119899 = 3)

growth led to significantly lower NO3-N content in KT204and TN86 Spraying Mo during the fast-growth period andsimultaneously spraying Mo and Gfo at the stage of maturityled to a significant decrease in NO3-N and NO2-N content inKT204 and TN86

35 Effects of Chemical Regulation on TSNAContents Auxinnaphthylacetic acid salicylic acid and malonic acid havebeen previously applied to decrease TSNA formation butthese may affect tobacco development and growth yield orquality [55 56] In this study we aimed to characterize achemical regulation strategy for decreasing TSNA precursors

so as to diminish TSNA formation in tobacco Yearly differ-ences in NO3-N NO2-N and TSNA contents in flue-curedtobacco were significant but TN and alkaloid levels werenot (Table 2) Regulatory treatments significantly affectedTN NO3-N alkaloid level and TSNA concentrations in flue-cured tobacco Varieties of burley tobacco were differentin TN NO3-N NO2-N alkaloid level and TSNA concen-trations and chemical regulation treatments significantlyaffected TN NO3-N and TSNA concentrations

As can be seen in Figure 5 spraying Mo during the fast-growth period and spraying Gfo at the stage of maturitydecreased TSNA concentrations in flue-cured tobacco but

Journal of Chemistry 7

CK

lowast

lowastlowast

lowastlowast

FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(a)

lowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

004

005

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(b)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

AVR

(gＧ

minus2Ｂminus1)

(c)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(d)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(e)

lowastlowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(f)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

40

AVR

(gＧ

minus2Ｂminus1)

(g)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(h)

Figure 3 Effects of Mo and Gfo treatments on NRA AVR GSA and SPRO in burley tobacco Error bars represent standard error (119899 = 3)NRA nitrate reductase activity AVR ammonia volatilization rate GSA glutamine synthetase activity SPRO total soluble protein content(andashd) KT204 (endashh) TN86 Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

8 Journal of Chemistry

Table 2 ANOVA comparison of the effects of regulatory treatments year and tobacco variety and their interactions on TN NO3-N NO2-Nalkaloid and TSNA concentrations in tobacco

Types Effect TN NO3-N NO2-N Alkaloid TSNAs DF

Flue-cured tobaccoYear (Y) 005ns 020lowast 001lowastlowast 160ns 004lowastlowast 1

Treatment (T) 901lowastlowast 1533lowastlowast 021ns 465lowast 719lowastlowast 3Year (Y) times Treatment (T) 854lowastlowast 5561lowastlowast 1371lowastlowast 573lowastlowast 3052lowastlowast 7

Burley tobaccoVariety (V) 097lowast 018lowast 1108lowastlowast 002lowastlowast 118lowast 1

Treatment (T) 359lowast 1934lowastlowast 097ns 028ns 1868lowastlowast 3Variety (V) times Treatment (T) 566lowastlowast 27557lowastlowast 2841lowastlowast 1314lowastlowast 27477lowastlowast 7

119865-values and significance levels are given (lowastlowast119901 lt 001 lowast119901 lt 005 and ns119901 ge 005)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(a)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowastlowastlowast

lowastlowast

0

500

1000

1500

2000

2500

3000

3500

N

3-N

cont

ent (

gＡminus

1D

W)

(b)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowast

0

1

2

3

4

N

2-N

cont

ent (

gＡminus

1D

W)

(c)M

-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1

2

3

4

Alk

aloi

d co

nten

t (

DW

)

(d)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

8

N

3-N

TN

()

(e)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(f)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

500

1000

1500

2000

2500

N

3-N

cont

ent (

gＡminus

1D

W)

(g)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowastlowast

00

05

10

15

20

N

2-N

cont

ent (

gＡminus

1D

W)

(h)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

2

4

6

Alk

aloi

d co

nten

t (

DW

)

(i)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

N

3-N

TN

()

(j)

Figure 4 Effects of treatments on total nitrogen content NO3-N content NO2-N content alkaloid content and NO3-NTN in burleytobacco NO3-NTN ratio of NO3-N and total nitrogen content (TN) Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicatesignificant difference at 001 or 005 respectively

the effect of spraying Mo during the fast-growth period wassignificantly different (119901 lt 005) Spraying Mo during thefast-growth period and spraying Mo and Gfo at maturityproduced the best results on TSNA concentrations among alltreatments in both 2014 and 2015 Spraying Mo during thefast-growth period could significantly reduce concentrations

of NNN NAB andNAT SprayingMo during the fast-growthperiod and spraying Gfo at the stage of maturity decreasedNNNNABNAT and total TSNA concentration in both 2014and 2015 respectively

TSNA accumulation in burley tobacco was much higherthan in flue-cured tobacco However effects of regulatory

Journal of Chemistry 9

2014

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

50

60

70

80

90

100

NN

N (n

gＡminus

1D

W)

(a)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

25

30

35

40

NAT

(ngＡminus

1D

W)

(b)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

20

25

30

35

40

NA

B (n

gＡminus

1D

W)

(c)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowastlowastlowast

lowastlowast

20

30

40

50

NN

K (n

gＡminus

1D

W)

(d)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast lowastlowast

lowastlowast

100

125

150

175

200

TSN

As (

ngＡminus

1D

W)

(e)2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

40

50

60

70

80

90

NN

N (n

gＡminus

1D

W)

(f)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

30

40

50

60

70

80

90

100

NAT

(ngＡminus

1D

W)

(g)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

10

15

20

25

30

NA

B (n

gＡminus

1D

W)

(h)

2015

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK20

25

30

35

40

45

50

55

60

NN

K (n

gＡminus

1D

W)

(i)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

80

120

160

200

240

TSN

As (

ngＡminus

1D

W)(j)

Figure 5 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in flue-cured tobacco Error bars indicatestandard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

treatment were more pronounced in burley tobacco Withinburley varieties the TSNA concentrations in KT204 werehigher than that in TN86 (Figures 6(a)ndash6(e)) Spraying ofModuring the fast-growth period led to significantly lowerNNNNAB and total TSNA concentrations in KT204 Sprayingof Gfo at maturity led to significant decreases in NNNNAT NNK and TSNA concentrations in TN86 The TSNA-regulating effects of the two treatments were optimized byspraying Mo during the fast-growth period and Gfo at thestage of maturity NNN NAT NAB NNK and total TSNAconcentration decreased in KT204 and TN86

36 Correlation Analysis Linear relationships betweenTSNAs alkaloids and NO3-N were significantly different(Figures 7(a)ndash7(c)) Total TSNA concentration in tobaccoincreased with increasing alkaloid and NO3-N contentespecially in burley tobacco The positive correlations

between TSNAs and their precursors were also reported byLewis et al [57] who suggested that NO3-N was a strongercontributing factor to higher TSNA levels than increasedalkaloid levels in burley tobacco

4 Conclusion

Nitrate was higher in burley tobacco than in flue-curedtobacco with both types showing peak nitrate content duringthe fast-growth period Under Mo treatment at the stage ofmaturity to avoid nitrate accumulation NRA LDM and DMin tobacco leaves increased SprayingMo in combinationwithGfo at the stage of maturity led to increased NRA and lowerGSA in tobacco which could help decrease nitrate and nitritecontent by increasing nitrogen loss via ammonia volatiliza-tion In summary spraying Mo during fast growth andspraying Mo with Gfo at the stage of maturity were effectivein reducing the formation of TSNAs

10 Journal of Chemistry

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

500

1000

1500

2000

NN

N (n

gＡminus

1D

W)

(a)

lowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

10

20

30

NA

B (n

gＡminus

1D

W)

(b)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

200

400

600

NAT

(ngＡminus

1D

W)

(c)

lowastlowastlowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(d)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1000

2000

3000

TSN

As (

ngＡminus

1D

W)

(e)

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

NN

N (n

gＡminus

1D

W)

(f)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

10

20

30

NA

B (n

gＡminus

1D

W)

(g)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

200

400

600

800N

AT (n

gＡminus

1D

W)

(h)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(i)

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

4000

TSN

As (

ngＡminus

1D

W)

(j)

Figure 6 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in burley tobacco varieties TN86 andKT204 Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

Burley tobacco

Flue-cured tobacco

R2 = 07220lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

3 4

Alkaloid content ( DW)

y = 15176x minus 35891

(a)

Burley tobacco

Flue-cured tobacco

R2 = 00557

0

2000

4000

TSN

As (

ngＡminus

1D

W)

321

N2-N content (g Ａminus1 DW)

y = 48834x + 17464

(b)

N3-N content (g Ａminus1 DW)3000200010000

Burley tobacco

Flue-cured tobacco

R2 = 07983lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

y = 121643x minus 28745602

(c)

Figure 7 Correlation analysis between TSNAs alkaloid NO2-N and NO3-N in tobacco Symbol lowastlowast indicates significant correlation at119901 lt 001

Journal of Chemistry 11

Gln

NR NiR

Glu Glu

Protein

Gln 2OG

NR NiRGS

TCA

TSNAs

Mo

NR Protein

NR GS

In fastgrowthperiod

At maturitystage

Leaf

Leaf

Nitratestorage

Nitrateaccumulation

Leaf

Nitratetransport

Glu Glu

Leaf

2OG

N（3

N（3

N（3

N3-N

N3-N

N3-N

N2-N

N2-N

Nx + lkaloid

MＩ + fo

Figure 8 Mechanisms for decreasing nitrate and TSNA concentrations in tobacco by spraying regulating chemicals Gfo glufosinate NRnitrate reductase NRA nitrate reductase activity NiR nitrite reductase GS glutamine synthetase GSA glutamine synthetase activity Glnglutamine Glu glutamate and OG oxaloacetate After sprayingMo on tobacco during the fast-growth period nitrate significantly decreasedwhile NRA and soluble protein content increasedThese decreased the amount of nitrate storage and promoted tobacco development duringthe fast-growth period After spraying Mo during the fast-growth stage and spraying Mo and Gfo at the stage of maturity NRA increasedand GSA decreased in tobacco which can significantly reduce nitrate accumulation and TSNA formation by nitrogen loss due to ammoniavolatilization

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank Editage (httpswwweditagecom) forEnglish language editing and publication support

References

[1] H R Burton N K Dye and L P Bush ldquoRelationship betweentobacco-specific nitrosamines and nitrite from different air-cured tobacco varietiesrdquo Journal of Agricultural and FoodChemistry vol 42 no 9 pp 2007ndash2011 1994

[2] B Siminszky L Gavilano S W Bowen and R E DeweyldquoConversion of nicotine to nornicotine in Nicotiana tabacumis mediated by CYP82E4 a cytochrome P450 monooxygenaserdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 41 pp 14919ndash14924 2005

[3] IARC ldquoSmokeless tobacco and some tobacco-specific N-nitrosamines monographs on the evaluation of carcinogenicrisks to humansrdquo National Publishing Group vol 89 Article ID1e592 2005

[4] H Shi R Wang and L P Bush ldquoThe relationships betweenTSNAs and their precursors in burley tobacco from differentregions and varieties JournalofFoodrdquoAgricultureEnvironmentvol 10 no 2 pp 1048ndash1052 2012

[5] H R Burton G H Childs Jr R A Andersen and P D Flem-ing ldquoChanges in chemical composition of Burley tobacco dur-ing senescence and curing 3 Tobacco-specific nitrosaminesrdquoJournal of Agricultural and Food Chemistry vol 37 no 2 pp426ndash430 1989

[6] H Shi R Wang L P Bush et al ldquoChanges in TSNA Contentsduring Tobacco Storage and the Effect of Temperature andNitrate Level on TSNA Formationrdquo Journal of Agricultural andFood Chemistry vol 61 pp 11588ndash11594 2013

[7] K Fytianos and P Zarogiannis ldquoNitrate and nitrite accumula-tion in fresh vegetables from Greecerdquo Bulletin of EnvironmentalContamination and Toxicology vol 62 no 2 pp 187ndash192 1999

[8] Y Y Wang P K Hsu and Y F Tsay ldquoUptake allocation andsignaling of nitraterdquo Trends in Plant Science vol 17 no 8 pp458ndash467 2012

[9] K S Reddy and R C Menary ldquoNitrate reductase and nitrateaccumulation in relation to nitrate toxicity in Boronia meg-astigmardquo Physiologia Plantarum vol 78 no 3 pp 430ndash4341990

[10] Y Han Q Liao Y Yu et al ldquoNitrate reutilization mechanismsin the tonoplast of two Brassica napus genotypes with differentnitrogen use efficiencyrdquoActa Physiologiae Plantarum vol 37 no2 2015

[11] H Kosaka K Imaizumi K Imai and I Tyuma ldquoStoichiometryof the reaction of oxyhemoglobin with nitriterdquo BBA - ProteinStructure vol 581 no 1 pp 184ndash188 1979

[12] P Santamaria ldquoNitrate in vegetables toxicity content intakeand EC regulationrdquo Journal of the Science of Food and Agricul-ture vol 86 no 1 pp 10ndash17 2006

[13] A T Diplock P J Aggett M Ashwell F Bornet E B FernandM B Roberfroid ldquoScientific concepts of functional foods in

Journal of Chemistry 7

CK

lowast

lowastlowast

lowastlowast

FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(a)

lowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

004

005

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(b)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

AVR

(gＧ

minus2Ｂminus1)

(c)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(d)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

NRA

(g

mＡminus

1Ｂminus1

FW)

(e)

lowastlowast

lowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo000

001

002

003

GSA

(m

ol m

Ａminus1Ｂminus1

FW)

(f)

lowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

10

20

30

40

AVR

(gＧ

minus2Ｂminus1)

(g)

lowastlowastlowastlowast

lowastlowast

CK FG-Mo M-Gfo M-M + fo0

50

100

150

200

SPRO

(mgＡminus

1FW

)

(h)

Figure 3 Effects of Mo and Gfo treatments on NRA AVR GSA and SPRO in burley tobacco Error bars represent standard error (119899 = 3)NRA nitrate reductase activity AVR ammonia volatilization rate GSA glutamine synthetase activity SPRO total soluble protein content(andashd) KT204 (endashh) TN86 Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

8 Journal of Chemistry

Table 2 ANOVA comparison of the effects of regulatory treatments year and tobacco variety and their interactions on TN NO3-N NO2-Nalkaloid and TSNA concentrations in tobacco

Types Effect TN NO3-N NO2-N Alkaloid TSNAs DF

Flue-cured tobaccoYear (Y) 005ns 020lowast 001lowastlowast 160ns 004lowastlowast 1

Treatment (T) 901lowastlowast 1533lowastlowast 021ns 465lowast 719lowastlowast 3Year (Y) times Treatment (T) 854lowastlowast 5561lowastlowast 1371lowastlowast 573lowastlowast 3052lowastlowast 7

Burley tobaccoVariety (V) 097lowast 018lowast 1108lowastlowast 002lowastlowast 118lowast 1

Treatment (T) 359lowast 1934lowastlowast 097ns 028ns 1868lowastlowast 3Variety (V) times Treatment (T) 566lowastlowast 27557lowastlowast 2841lowastlowast 1314lowastlowast 27477lowastlowast 7

119865-values and significance levels are given (lowastlowast119901 lt 001 lowast119901 lt 005 and ns119901 ge 005)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(a)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowastlowastlowast

lowastlowast

0

500

1000

1500

2000

2500

3000

3500

N

3-N

cont

ent (

gＡminus

1D

W)

(b)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowast

0

1

2

3

4

N

2-N

cont

ent (

gＡminus

1D

W)

(c)M

-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1

2

3

4

Alk

aloi

d co

nten

t (

DW

)

(d)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

8

N

3-N

TN

()

(e)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(f)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

500

1000

1500

2000

2500

N

3-N

cont

ent (

gＡminus

1D

W)

(g)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowastlowast

00

05

10

15

20

N

2-N

cont

ent (

gＡminus

1D

W)

(h)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

2

4

6

Alk

aloi

d co

nten

t (

DW

)

(i)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

N

3-N

TN

()

(j)

Figure 4 Effects of treatments on total nitrogen content NO3-N content NO2-N content alkaloid content and NO3-NTN in burleytobacco NO3-NTN ratio of NO3-N and total nitrogen content (TN) Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicatesignificant difference at 001 or 005 respectively

the effect of spraying Mo during the fast-growth period wassignificantly different (119901 lt 005) Spraying Mo during thefast-growth period and spraying Mo and Gfo at maturityproduced the best results on TSNA concentrations among alltreatments in both 2014 and 2015 Spraying Mo during thefast-growth period could significantly reduce concentrations

of NNN NAB andNAT SprayingMo during the fast-growthperiod and spraying Gfo at the stage of maturity decreasedNNNNABNAT and total TSNA concentration in both 2014and 2015 respectively

TSNA accumulation in burley tobacco was much higherthan in flue-cured tobacco However effects of regulatory

Journal of Chemistry 9

2014

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

50

60

70

80

90

100

NN

N (n

gＡminus

1D

W)

(a)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

25

30

35

40

NAT

(ngＡminus

1D

W)

(b)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

20

25

30

35

40

NA

B (n

gＡminus

1D

W)

(c)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowastlowastlowast

lowastlowast

20

30

40

50

NN

K (n

gＡminus

1D

W)

(d)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast lowastlowast

lowastlowast

100

125

150

175

200

TSN

As (

ngＡminus

1D

W)

(e)2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

40

50

60

70

80

90

NN

N (n

gＡminus

1D

W)

(f)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

30

40

50

60

70

80

90

100

NAT

(ngＡminus

1D

W)

(g)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

10

15

20

25

30

NA

B (n

gＡminus

1D

W)

(h)

2015

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK20

25

30

35

40

45

50

55

60

NN

K (n

gＡminus

1D

W)

(i)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

80

120

160

200

240

TSN

As (

ngＡminus

1D

W)(j)

Figure 5 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in flue-cured tobacco Error bars indicatestandard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

treatment were more pronounced in burley tobacco Withinburley varieties the TSNA concentrations in KT204 werehigher than that in TN86 (Figures 6(a)ndash6(e)) Spraying ofModuring the fast-growth period led to significantly lowerNNNNAB and total TSNA concentrations in KT204 Sprayingof Gfo at maturity led to significant decreases in NNNNAT NNK and TSNA concentrations in TN86 The TSNA-regulating effects of the two treatments were optimized byspraying Mo during the fast-growth period and Gfo at thestage of maturity NNN NAT NAB NNK and total TSNAconcentration decreased in KT204 and TN86

36 Correlation Analysis Linear relationships betweenTSNAs alkaloids and NO3-N were significantly different(Figures 7(a)ndash7(c)) Total TSNA concentration in tobaccoincreased with increasing alkaloid and NO3-N contentespecially in burley tobacco The positive correlations

between TSNAs and their precursors were also reported byLewis et al [57] who suggested that NO3-N was a strongercontributing factor to higher TSNA levels than increasedalkaloid levels in burley tobacco

4 Conclusion

Nitrate was higher in burley tobacco than in flue-curedtobacco with both types showing peak nitrate content duringthe fast-growth period Under Mo treatment at the stage ofmaturity to avoid nitrate accumulation NRA LDM and DMin tobacco leaves increased SprayingMo in combinationwithGfo at the stage of maturity led to increased NRA and lowerGSA in tobacco which could help decrease nitrate and nitritecontent by increasing nitrogen loss via ammonia volatiliza-tion In summary spraying Mo during fast growth andspraying Mo with Gfo at the stage of maturity were effectivein reducing the formation of TSNAs

10 Journal of Chemistry

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

500

1000

1500

2000

NN

N (n

gＡminus

1D

W)

(a)

lowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

10

20

30

NA

B (n

gＡminus

1D

W)

(b)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

200

400

600

NAT

(ngＡminus

1D

W)

(c)

lowastlowastlowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(d)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1000

2000

3000

TSN

As (

ngＡminus

1D

W)

(e)

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

NN

N (n

gＡminus

1D

W)

(f)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

10

20

30

NA

B (n

gＡminus

1D

W)

(g)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

200

400

600

800N

AT (n

gＡminus

1D

W)

(h)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(i)

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

4000

TSN

As (

ngＡminus

1D

W)

(j)

Figure 6 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in burley tobacco varieties TN86 andKT204 Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

Burley tobacco

Flue-cured tobacco

R2 = 07220lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

3 4

Alkaloid content ( DW)

y = 15176x minus 35891

(a)

Burley tobacco

Flue-cured tobacco

R2 = 00557

0

2000

4000

TSN

As (

ngＡminus

1D

W)

321

N2-N content (g Ａminus1 DW)

y = 48834x + 17464

(b)

N3-N content (g Ａminus1 DW)3000200010000

Burley tobacco

Flue-cured tobacco

R2 = 07983lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

y = 121643x minus 28745602

(c)

Figure 7 Correlation analysis between TSNAs alkaloid NO2-N and NO3-N in tobacco Symbol lowastlowast indicates significant correlation at119901 lt 001

Journal of Chemistry 11

Gln

NR NiR

Glu Glu

Protein

Gln 2OG

NR NiRGS

TCA

TSNAs

Mo

NR Protein

NR GS

In fastgrowthperiod

At maturitystage

Leaf

Leaf

Nitratestorage

Nitrateaccumulation

Leaf

Nitratetransport

Glu Glu

Leaf

2OG

N（3

N（3

N（3

N3-N

N3-N

N3-N

N2-N

N2-N

Nx + lkaloid

MＩ + fo

Figure 8 Mechanisms for decreasing nitrate and TSNA concentrations in tobacco by spraying regulating chemicals Gfo glufosinate NRnitrate reductase NRA nitrate reductase activity NiR nitrite reductase GS glutamine synthetase GSA glutamine synthetase activity Glnglutamine Glu glutamate and OG oxaloacetate After sprayingMo on tobacco during the fast-growth period nitrate significantly decreasedwhile NRA and soluble protein content increasedThese decreased the amount of nitrate storage and promoted tobacco development duringthe fast-growth period After spraying Mo during the fast-growth stage and spraying Mo and Gfo at the stage of maturity NRA increasedand GSA decreased in tobacco which can significantly reduce nitrate accumulation and TSNA formation by nitrogen loss due to ammoniavolatilization

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank Editage (httpswwweditagecom) forEnglish language editing and publication support

References

[1] H R Burton N K Dye and L P Bush ldquoRelationship betweentobacco-specific nitrosamines and nitrite from different air-cured tobacco varietiesrdquo Journal of Agricultural and FoodChemistry vol 42 no 9 pp 2007ndash2011 1994

[2] B Siminszky L Gavilano S W Bowen and R E DeweyldquoConversion of nicotine to nornicotine in Nicotiana tabacumis mediated by CYP82E4 a cytochrome P450 monooxygenaserdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 41 pp 14919ndash14924 2005

[3] IARC ldquoSmokeless tobacco and some tobacco-specific N-nitrosamines monographs on the evaluation of carcinogenicrisks to humansrdquo National Publishing Group vol 89 Article ID1e592 2005

[4] H Shi R Wang and L P Bush ldquoThe relationships betweenTSNAs and their precursors in burley tobacco from differentregions and varieties JournalofFoodrdquoAgricultureEnvironmentvol 10 no 2 pp 1048ndash1052 2012

[5] H R Burton G H Childs Jr R A Andersen and P D Flem-ing ldquoChanges in chemical composition of Burley tobacco dur-ing senescence and curing 3 Tobacco-specific nitrosaminesrdquoJournal of Agricultural and Food Chemistry vol 37 no 2 pp426ndash430 1989

[6] H Shi R Wang L P Bush et al ldquoChanges in TSNA Contentsduring Tobacco Storage and the Effect of Temperature andNitrate Level on TSNA Formationrdquo Journal of Agricultural andFood Chemistry vol 61 pp 11588ndash11594 2013

[7] K Fytianos and P Zarogiannis ldquoNitrate and nitrite accumula-tion in fresh vegetables from Greecerdquo Bulletin of EnvironmentalContamination and Toxicology vol 62 no 2 pp 187ndash192 1999

[8] Y Y Wang P K Hsu and Y F Tsay ldquoUptake allocation andsignaling of nitraterdquo Trends in Plant Science vol 17 no 8 pp458ndash467 2012

[9] K S Reddy and R C Menary ldquoNitrate reductase and nitrateaccumulation in relation to nitrate toxicity in Boronia meg-astigmardquo Physiologia Plantarum vol 78 no 3 pp 430ndash4341990

[10] Y Han Q Liao Y Yu et al ldquoNitrate reutilization mechanismsin the tonoplast of two Brassica napus genotypes with differentnitrogen use efficiencyrdquoActa Physiologiae Plantarum vol 37 no2 2015

[11] H Kosaka K Imaizumi K Imai and I Tyuma ldquoStoichiometryof the reaction of oxyhemoglobin with nitriterdquo BBA - ProteinStructure vol 581 no 1 pp 184ndash188 1979

[12] P Santamaria ldquoNitrate in vegetables toxicity content intakeand EC regulationrdquo Journal of the Science of Food and Agricul-ture vol 86 no 1 pp 10ndash17 2006

[13] A T Diplock P J Aggett M Ashwell F Bornet E B FernandM B Roberfroid ldquoScientific concepts of functional foods in

8 Journal of Chemistry

Table 2 ANOVA comparison of the effects of regulatory treatments year and tobacco variety and their interactions on TN NO3-N NO2-Nalkaloid and TSNA concentrations in tobacco

Types Effect TN NO3-N NO2-N Alkaloid TSNAs DF

Flue-cured tobaccoYear (Y) 005ns 020lowast 001lowastlowast 160ns 004lowastlowast 1

Treatment (T) 901lowastlowast 1533lowastlowast 021ns 465lowast 719lowastlowast 3Year (Y) times Treatment (T) 854lowastlowast 5561lowastlowast 1371lowastlowast 573lowastlowast 3052lowastlowast 7

Burley tobaccoVariety (V) 097lowast 018lowast 1108lowastlowast 002lowastlowast 118lowast 1

Treatment (T) 359lowast 1934lowastlowast 097ns 028ns 1868lowastlowast 3Variety (V) times Treatment (T) 566lowastlowast 27557lowastlowast 2841lowastlowast 1314lowastlowast 27477lowastlowast 7

119865-values and significance levels are given (lowastlowast119901 lt 001 lowast119901 lt 005 and ns119901 ge 005)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(a)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowastlowastlowast

lowastlowast

0

500

1000

1500

2000

2500

3000

3500

N

3-N

cont

ent (

gＡminus

1D

W)

(b)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowast lowast

0

1

2

3

4

N

2-N

cont

ent (

gＡminus

1D

W)

(c)M

-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1

2

3

4

Alk

aloi

d co

nten

t (

DW

)

(d)

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

8

N

3-N

TN

()

(e)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

0

2

4

6

Tota

l nitr

ogen

cont

ent (

D

W)

(f)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

500

1000

1500

2000

2500

N

3-N

cont

ent (

gＡminus

1D

W)

(g)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowastlowast

00

05

10

15

20

N

2-N

cont

ent (

gＡminus

1D

W)

(h)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

2

4

6

Alk

aloi

d co

nten

t (

DW

)

(i)

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

lowastlowast

lowastlowast

lowastlowast

0

2

4

6

N

3-N

TN

()

(j)

Figure 4 Effects of treatments on total nitrogen content NO3-N content NO2-N content alkaloid content and NO3-NTN in burleytobacco NO3-NTN ratio of NO3-N and total nitrogen content (TN) Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicatesignificant difference at 001 or 005 respectively

the effect of spraying Mo during the fast-growth period wassignificantly different (119901 lt 005) Spraying Mo during thefast-growth period and spraying Mo and Gfo at maturityproduced the best results on TSNA concentrations among alltreatments in both 2014 and 2015 Spraying Mo during thefast-growth period could significantly reduce concentrations

of NNN NAB andNAT SprayingMo during the fast-growthperiod and spraying Gfo at the stage of maturity decreasedNNNNABNAT and total TSNA concentration in both 2014and 2015 respectively

TSNA accumulation in burley tobacco was much higherthan in flue-cured tobacco However effects of regulatory

Journal of Chemistry 9

2014

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

50

60

70

80

90

100

NN

N (n

gＡminus

1D

W)

(a)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

25

30

35

40

NAT

(ngＡminus

1D

W)

(b)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

20

25

30

35

40

NA

B (n

gＡminus

1D

W)

(c)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowastlowastlowast

lowastlowast

20

30

40

50

NN

K (n

gＡminus

1D

W)

(d)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast lowastlowast

lowastlowast

100

125

150

175

200

TSN

As (

ngＡminus

1D

W)

(e)2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

40

50

60

70

80

90

NN

N (n

gＡminus

1D

W)

(f)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

30

40

50

60

70

80

90

100

NAT

(ngＡminus

1D

W)

(g)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

10

15

20

25

30

NA

B (n

gＡminus

1D

W)

(h)

2015

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK20

25

30

35

40

45

50

55

60

NN

K (n

gＡminus

1D

W)

(i)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

80

120

160

200

240

TSN

As (

ngＡminus

1D

W)(j)

Figure 5 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in flue-cured tobacco Error bars indicatestandard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

treatment were more pronounced in burley tobacco Withinburley varieties the TSNA concentrations in KT204 werehigher than that in TN86 (Figures 6(a)ndash6(e)) Spraying ofModuring the fast-growth period led to significantly lowerNNNNAB and total TSNA concentrations in KT204 Sprayingof Gfo at maturity led to significant decreases in NNNNAT NNK and TSNA concentrations in TN86 The TSNA-regulating effects of the two treatments were optimized byspraying Mo during the fast-growth period and Gfo at thestage of maturity NNN NAT NAB NNK and total TSNAconcentration decreased in KT204 and TN86

36 Correlation Analysis Linear relationships betweenTSNAs alkaloids and NO3-N were significantly different(Figures 7(a)ndash7(c)) Total TSNA concentration in tobaccoincreased with increasing alkaloid and NO3-N contentespecially in burley tobacco The positive correlations

between TSNAs and their precursors were also reported byLewis et al [57] who suggested that NO3-N was a strongercontributing factor to higher TSNA levels than increasedalkaloid levels in burley tobacco

4 Conclusion

Nitrate was higher in burley tobacco than in flue-curedtobacco with both types showing peak nitrate content duringthe fast-growth period Under Mo treatment at the stage ofmaturity to avoid nitrate accumulation NRA LDM and DMin tobacco leaves increased SprayingMo in combinationwithGfo at the stage of maturity led to increased NRA and lowerGSA in tobacco which could help decrease nitrate and nitritecontent by increasing nitrogen loss via ammonia volatiliza-tion In summary spraying Mo during fast growth andspraying Mo with Gfo at the stage of maturity were effectivein reducing the formation of TSNAs

10 Journal of Chemistry

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

500

1000

1500

2000

NN

N (n

gＡminus

1D

W)

(a)

lowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

10

20

30

NA

B (n

gＡminus

1D

W)

(b)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

200

400

600

NAT

(ngＡminus

1D

W)

(c)

lowastlowastlowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(d)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1000

2000

3000

TSN

As (

ngＡminus

1D

W)

(e)

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

NN

N (n

gＡminus

1D

W)

(f)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

10

20

30

NA

B (n

gＡminus

1D

W)

(g)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

200

400

600

800N

AT (n

gＡminus

1D

W)

(h)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(i)

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

4000

TSN

As (

ngＡminus

1D

W)

(j)

Figure 6 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in burley tobacco varieties TN86 andKT204 Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

Burley tobacco

Flue-cured tobacco

R2 = 07220lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

3 4

Alkaloid content ( DW)

y = 15176x minus 35891

(a)

Burley tobacco

Flue-cured tobacco

R2 = 00557

0

2000

4000

TSN

As (

ngＡminus

1D

W)

321

N2-N content (g Ａminus1 DW)

y = 48834x + 17464

(b)

N3-N content (g Ａminus1 DW)3000200010000

Burley tobacco

Flue-cured tobacco

R2 = 07983lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

y = 121643x minus 28745602

(c)

Figure 7 Correlation analysis between TSNAs alkaloid NO2-N and NO3-N in tobacco Symbol lowastlowast indicates significant correlation at119901 lt 001

Journal of Chemistry 11

Gln

NR NiR

Glu Glu

Protein

Gln 2OG

NR NiRGS

TCA

TSNAs

Mo

NR Protein

NR GS

In fastgrowthperiod

At maturitystage

Leaf

Leaf

Nitratestorage

Nitrateaccumulation

Leaf

Nitratetransport

Glu Glu

Leaf

2OG

N（3

N（3

N（3

N3-N

N3-N

N3-N

N2-N

N2-N

Nx + lkaloid

MＩ + fo

Figure 8 Mechanisms for decreasing nitrate and TSNA concentrations in tobacco by spraying regulating chemicals Gfo glufosinate NRnitrate reductase NRA nitrate reductase activity NiR nitrite reductase GS glutamine synthetase GSA glutamine synthetase activity Glnglutamine Glu glutamate and OG oxaloacetate After sprayingMo on tobacco during the fast-growth period nitrate significantly decreasedwhile NRA and soluble protein content increasedThese decreased the amount of nitrate storage and promoted tobacco development duringthe fast-growth period After spraying Mo during the fast-growth stage and spraying Mo and Gfo at the stage of maturity NRA increasedand GSA decreased in tobacco which can significantly reduce nitrate accumulation and TSNA formation by nitrogen loss due to ammoniavolatilization

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank Editage (httpswwweditagecom) forEnglish language editing and publication support

References

[1] H R Burton N K Dye and L P Bush ldquoRelationship betweentobacco-specific nitrosamines and nitrite from different air-cured tobacco varietiesrdquo Journal of Agricultural and FoodChemistry vol 42 no 9 pp 2007ndash2011 1994

[2] B Siminszky L Gavilano S W Bowen and R E DeweyldquoConversion of nicotine to nornicotine in Nicotiana tabacumis mediated by CYP82E4 a cytochrome P450 monooxygenaserdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 41 pp 14919ndash14924 2005

[3] IARC ldquoSmokeless tobacco and some tobacco-specific N-nitrosamines monographs on the evaluation of carcinogenicrisks to humansrdquo National Publishing Group vol 89 Article ID1e592 2005

[4] H Shi R Wang and L P Bush ldquoThe relationships betweenTSNAs and their precursors in burley tobacco from differentregions and varieties JournalofFoodrdquoAgricultureEnvironmentvol 10 no 2 pp 1048ndash1052 2012

[5] H R Burton G H Childs Jr R A Andersen and P D Flem-ing ldquoChanges in chemical composition of Burley tobacco dur-ing senescence and curing 3 Tobacco-specific nitrosaminesrdquoJournal of Agricultural and Food Chemistry vol 37 no 2 pp426ndash430 1989

[6] H Shi R Wang L P Bush et al ldquoChanges in TSNA Contentsduring Tobacco Storage and the Effect of Temperature andNitrate Level on TSNA Formationrdquo Journal of Agricultural andFood Chemistry vol 61 pp 11588ndash11594 2013

[7] K Fytianos and P Zarogiannis ldquoNitrate and nitrite accumula-tion in fresh vegetables from Greecerdquo Bulletin of EnvironmentalContamination and Toxicology vol 62 no 2 pp 187ndash192 1999

[8] Y Y Wang P K Hsu and Y F Tsay ldquoUptake allocation andsignaling of nitraterdquo Trends in Plant Science vol 17 no 8 pp458ndash467 2012

[9] K S Reddy and R C Menary ldquoNitrate reductase and nitrateaccumulation in relation to nitrate toxicity in Boronia meg-astigmardquo Physiologia Plantarum vol 78 no 3 pp 430ndash4341990

[10] Y Han Q Liao Y Yu et al ldquoNitrate reutilization mechanismsin the tonoplast of two Brassica napus genotypes with differentnitrogen use efficiencyrdquoActa Physiologiae Plantarum vol 37 no2 2015

[11] H Kosaka K Imaizumi K Imai and I Tyuma ldquoStoichiometryof the reaction of oxyhemoglobin with nitriterdquo BBA - ProteinStructure vol 581 no 1 pp 184ndash188 1979

[12] P Santamaria ldquoNitrate in vegetables toxicity content intakeand EC regulationrdquo Journal of the Science of Food and Agricul-ture vol 86 no 1 pp 10ndash17 2006

[13] A T Diplock P J Aggett M Ashwell F Bornet E B FernandM B Roberfroid ldquoScientific concepts of functional foods in

Journal of Chemistry 9

2014

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

50

60

70

80

90

100

NN

N (n

gＡminus

1D

W)

(a)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

25

30

35

40

NAT

(ngＡminus

1D

W)

(b)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

20

25

30

35

40

NA

B (n

gＡminus

1D

W)

(c)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowastlowastlowast

lowastlowast

20

30

40

50

NN

K (n

gＡminus

1D

W)

(d)

2014

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast lowastlowast

lowastlowast

100

125

150

175

200

TSN

As (

ngＡminus

1D

W)

(e)2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

40

50

60

70

80

90

NN

N (n

gＡminus

1D

W)

(f)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

30

40

50

60

70

80

90

100

NAT

(ngＡminus

1D

W)

(g)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowast

lowastlowast

10

15

20

25

30

NA

B (n

gＡminus

1D

W)

(h)

2015

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK20

25

30

35

40

45

50

55

60

NN

K (n

gＡminus

1D

W)

(i)

2015

M-M

+

fo

M-G

fo

FG-M

o

CK

lowastlowast

lowastlowastlowastlowast

80

120

160

200

240

TSN

As (

ngＡminus

1D

W)(j)

Figure 5 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in flue-cured tobacco Error bars indicatestandard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

treatment were more pronounced in burley tobacco Withinburley varieties the TSNA concentrations in KT204 werehigher than that in TN86 (Figures 6(a)ndash6(e)) Spraying ofModuring the fast-growth period led to significantly lowerNNNNAB and total TSNA concentrations in KT204 Sprayingof Gfo at maturity led to significant decreases in NNNNAT NNK and TSNA concentrations in TN86 The TSNA-regulating effects of the two treatments were optimized byspraying Mo during the fast-growth period and Gfo at thestage of maturity NNN NAT NAB NNK and total TSNAconcentration decreased in KT204 and TN86

36 Correlation Analysis Linear relationships betweenTSNAs alkaloids and NO3-N were significantly different(Figures 7(a)ndash7(c)) Total TSNA concentration in tobaccoincreased with increasing alkaloid and NO3-N contentespecially in burley tobacco The positive correlations

between TSNAs and their precursors were also reported byLewis et al [57] who suggested that NO3-N was a strongercontributing factor to higher TSNA levels than increasedalkaloid levels in burley tobacco

4 Conclusion

Nitrate was higher in burley tobacco than in flue-curedtobacco with both types showing peak nitrate content duringthe fast-growth period Under Mo treatment at the stage ofmaturity to avoid nitrate accumulation NRA LDM and DMin tobacco leaves increased SprayingMo in combinationwithGfo at the stage of maturity led to increased NRA and lowerGSA in tobacco which could help decrease nitrate and nitritecontent by increasing nitrogen loss via ammonia volatiliza-tion In summary spraying Mo during fast growth andspraying Mo with Gfo at the stage of maturity were effectivein reducing the formation of TSNAs

10 Journal of Chemistry

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

500

1000

1500

2000

NN

N (n

gＡminus

1D

W)

(a)

lowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

10

20

30

NA

B (n

gＡminus

1D

W)

(b)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

200

400

600

NAT

(ngＡminus

1D

W)

(c)

lowastlowastlowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(d)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1000

2000

3000

TSN

As (

ngＡminus

1D

W)

(e)

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

NN

N (n

gＡminus

1D

W)

(f)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

10

20

30

NA

B (n

gＡminus

1D

W)

(g)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

200

400

600

800N

AT (n

gＡminus

1D

W)

(h)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(i)

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

4000

TSN

As (

ngＡminus

1D

W)

(j)

Figure 6 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in burley tobacco varieties TN86 andKT204 Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

Burley tobacco

Flue-cured tobacco

R2 = 07220lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

3 4

Alkaloid content ( DW)

y = 15176x minus 35891

(a)

Burley tobacco

Flue-cured tobacco

R2 = 00557

0

2000

4000

TSN

As (

ngＡminus

1D

W)

321

N2-N content (g Ａminus1 DW)

y = 48834x + 17464

(b)

N3-N content (g Ａminus1 DW)3000200010000

Burley tobacco

Flue-cured tobacco

R2 = 07983lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

y = 121643x minus 28745602

(c)

Figure 7 Correlation analysis between TSNAs alkaloid NO2-N and NO3-N in tobacco Symbol lowastlowast indicates significant correlation at119901 lt 001

Journal of Chemistry 11

Gln

NR NiR

Glu Glu

Protein

Gln 2OG

NR NiRGS

TCA

TSNAs

Mo

NR Protein

NR GS

In fastgrowthperiod

At maturitystage

Leaf

Leaf

Nitratestorage

Nitrateaccumulation

Leaf

Nitratetransport

Glu Glu

Leaf

2OG

N（3

N（3

N（3

N3-N

N3-N

N3-N

N2-N

N2-N

Nx + lkaloid

MＩ + fo

Figure 8 Mechanisms for decreasing nitrate and TSNA concentrations in tobacco by spraying regulating chemicals Gfo glufosinate NRnitrate reductase NRA nitrate reductase activity NiR nitrite reductase GS glutamine synthetase GSA glutamine synthetase activity Glnglutamine Glu glutamate and OG oxaloacetate After sprayingMo on tobacco during the fast-growth period nitrate significantly decreasedwhile NRA and soluble protein content increasedThese decreased the amount of nitrate storage and promoted tobacco development duringthe fast-growth period After spraying Mo during the fast-growth stage and spraying Mo and Gfo at the stage of maturity NRA increasedand GSA decreased in tobacco which can significantly reduce nitrate accumulation and TSNA formation by nitrogen loss due to ammoniavolatilization

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank Editage (httpswwweditagecom) forEnglish language editing and publication support

References

[1] H R Burton N K Dye and L P Bush ldquoRelationship betweentobacco-specific nitrosamines and nitrite from different air-cured tobacco varietiesrdquo Journal of Agricultural and FoodChemistry vol 42 no 9 pp 2007ndash2011 1994

[2] B Siminszky L Gavilano S W Bowen and R E DeweyldquoConversion of nicotine to nornicotine in Nicotiana tabacumis mediated by CYP82E4 a cytochrome P450 monooxygenaserdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 41 pp 14919ndash14924 2005

[3] IARC ldquoSmokeless tobacco and some tobacco-specific N-nitrosamines monographs on the evaluation of carcinogenicrisks to humansrdquo National Publishing Group vol 89 Article ID1e592 2005

[4] H Shi R Wang and L P Bush ldquoThe relationships betweenTSNAs and their precursors in burley tobacco from differentregions and varieties JournalofFoodrdquoAgricultureEnvironmentvol 10 no 2 pp 1048ndash1052 2012

[5] H R Burton G H Childs Jr R A Andersen and P D Flem-ing ldquoChanges in chemical composition of Burley tobacco dur-ing senescence and curing 3 Tobacco-specific nitrosaminesrdquoJournal of Agricultural and Food Chemistry vol 37 no 2 pp426ndash430 1989

[6] H Shi R Wang L P Bush et al ldquoChanges in TSNA Contentsduring Tobacco Storage and the Effect of Temperature andNitrate Level on TSNA Formationrdquo Journal of Agricultural andFood Chemistry vol 61 pp 11588ndash11594 2013

[7] K Fytianos and P Zarogiannis ldquoNitrate and nitrite accumula-tion in fresh vegetables from Greecerdquo Bulletin of EnvironmentalContamination and Toxicology vol 62 no 2 pp 187ndash192 1999

[8] Y Y Wang P K Hsu and Y F Tsay ldquoUptake allocation andsignaling of nitraterdquo Trends in Plant Science vol 17 no 8 pp458ndash467 2012

[9] K S Reddy and R C Menary ldquoNitrate reductase and nitrateaccumulation in relation to nitrate toxicity in Boronia meg-astigmardquo Physiologia Plantarum vol 78 no 3 pp 430ndash4341990

[10] Y Han Q Liao Y Yu et al ldquoNitrate reutilization mechanismsin the tonoplast of two Brassica napus genotypes with differentnitrogen use efficiencyrdquoActa Physiologiae Plantarum vol 37 no2 2015

[11] H Kosaka K Imaizumi K Imai and I Tyuma ldquoStoichiometryof the reaction of oxyhemoglobin with nitriterdquo BBA - ProteinStructure vol 581 no 1 pp 184ndash188 1979

[12] P Santamaria ldquoNitrate in vegetables toxicity content intakeand EC regulationrdquo Journal of the Science of Food and Agricul-ture vol 86 no 1 pp 10ndash17 2006

[13] A T Diplock P J Aggett M Ashwell F Bornet E B FernandM B Roberfroid ldquoScientific concepts of functional foods in

10 Journal of Chemistry

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

500

1000

1500

2000

NN

N (n

gＡminus

1D

W)

(a)

lowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

10

20

30

NA

B (n

gＡminus

1D

W)

(b)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

200

400

600

NAT

(ngＡminus

1D

W)

(c)

lowastlowastlowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(d)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

TN86

0

1000

2000

3000

TSN

As (

ngＡminus

1D

W)

(e)

lowastlowast

lowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

NN

N (n

gＡminus

1D

W)

(f)

lowastlowast

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

10

20

30

NA

B (n

gＡminus

1D

W)

(g)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

200

400

600

800N

AT (n

gＡminus

1D

W)

(h)

lowastlowastlowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

50

100

150

200

NN

K (n

gＡminus

1D

W)

(i)

lowastlowastlowastlowast

lowastlowast

M-M

+

fo

M-G

fo

FG-M

o

CK

KT204

0

1000

2000

3000

4000

TSN

As (

ngＡminus

1D

W)

(j)

Figure 6 Effects of chemical regulation on NNN NAB NAT NNK and total TSNA concentration in burley tobacco varieties TN86 andKT204 Error bars represent standard error (119899 = 3) Symbols lowastlowast and lowast indicate significant difference at 001 or 005 respectively

Burley tobacco

Flue-cured tobacco

R2 = 07220lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

3 4

Alkaloid content ( DW)

y = 15176x minus 35891

(a)

Burley tobacco

Flue-cured tobacco

R2 = 00557

0

2000

4000

TSN

As (

ngＡminus

1D

W)

321

N2-N content (g Ａminus1 DW)

y = 48834x + 17464

(b)

N3-N content (g Ａminus1 DW)3000200010000

Burley tobacco

Flue-cured tobacco

R2 = 07983lowastlowast

0

2000

4000

TSN

As (

ngＡminus

1D

W)

y = 121643x minus 28745602

(c)

Figure 7 Correlation analysis between TSNAs alkaloid NO2-N and NO3-N in tobacco Symbol lowastlowast indicates significant correlation at119901 lt 001

Journal of Chemistry 11

Gln

NR NiR

Glu Glu

Protein

Gln 2OG

NR NiRGS

TCA

TSNAs

Mo

NR Protein

NR GS

In fastgrowthperiod

At maturitystage

Leaf

Leaf

Nitratestorage

Nitrateaccumulation

Leaf

Nitratetransport

Glu Glu

Leaf

2OG

N（3

N（3

N（3

N3-N

N3-N

N3-N

N2-N

N2-N

Nx + lkaloid

MＩ + fo

Figure 8 Mechanisms for decreasing nitrate and TSNA concentrations in tobacco by spraying regulating chemicals Gfo glufosinate NRnitrate reductase NRA nitrate reductase activity NiR nitrite reductase GS glutamine synthetase GSA glutamine synthetase activity Glnglutamine Glu glutamate and OG oxaloacetate After sprayingMo on tobacco during the fast-growth period nitrate significantly decreasedwhile NRA and soluble protein content increasedThese decreased the amount of nitrate storage and promoted tobacco development duringthe fast-growth period After spraying Mo during the fast-growth stage and spraying Mo and Gfo at the stage of maturity NRA increasedand GSA decreased in tobacco which can significantly reduce nitrate accumulation and TSNA formation by nitrogen loss due to ammoniavolatilization

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank Editage (httpswwweditagecom) forEnglish language editing and publication support

References

[1] H R Burton N K Dye and L P Bush ldquoRelationship betweentobacco-specific nitrosamines and nitrite from different air-cured tobacco varietiesrdquo Journal of Agricultural and FoodChemistry vol 42 no 9 pp 2007ndash2011 1994

[2] B Siminszky L Gavilano S W Bowen and R E DeweyldquoConversion of nicotine to nornicotine in Nicotiana tabacumis mediated by CYP82E4 a cytochrome P450 monooxygenaserdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 41 pp 14919ndash14924 2005

[3] IARC ldquoSmokeless tobacco and some tobacco-specific N-nitrosamines monographs on the evaluation of carcinogenicrisks to humansrdquo National Publishing Group vol 89 Article ID1e592 2005

[4] H Shi R Wang and L P Bush ldquoThe relationships betweenTSNAs and their precursors in burley tobacco from differentregions and varieties JournalofFoodrdquoAgricultureEnvironmentvol 10 no 2 pp 1048ndash1052 2012

[5] H R Burton G H Childs Jr R A Andersen and P D Flem-ing ldquoChanges in chemical composition of Burley tobacco dur-ing senescence and curing 3 Tobacco-specific nitrosaminesrdquoJournal of Agricultural and Food Chemistry vol 37 no 2 pp426ndash430 1989

[6] H Shi R Wang L P Bush et al ldquoChanges in TSNA Contentsduring Tobacco Storage and the Effect of Temperature andNitrate Level on TSNA Formationrdquo Journal of Agricultural andFood Chemistry vol 61 pp 11588ndash11594 2013

[7] K Fytianos and P Zarogiannis ldquoNitrate and nitrite accumula-tion in fresh vegetables from Greecerdquo Bulletin of EnvironmentalContamination and Toxicology vol 62 no 2 pp 187ndash192 1999

[8] Y Y Wang P K Hsu and Y F Tsay ldquoUptake allocation andsignaling of nitraterdquo Trends in Plant Science vol 17 no 8 pp458ndash467 2012

[9] K S Reddy and R C Menary ldquoNitrate reductase and nitrateaccumulation in relation to nitrate toxicity in Boronia meg-astigmardquo Physiologia Plantarum vol 78 no 3 pp 430ndash4341990

[10] Y Han Q Liao Y Yu et al ldquoNitrate reutilization mechanismsin the tonoplast of two Brassica napus genotypes with differentnitrogen use efficiencyrdquoActa Physiologiae Plantarum vol 37 no2 2015

[11] H Kosaka K Imaizumi K Imai and I Tyuma ldquoStoichiometryof the reaction of oxyhemoglobin with nitriterdquo BBA - ProteinStructure vol 581 no 1 pp 184ndash188 1979

[12] P Santamaria ldquoNitrate in vegetables toxicity content intakeand EC regulationrdquo Journal of the Science of Food and Agricul-ture vol 86 no 1 pp 10ndash17 2006

[13] A T Diplock P J Aggett M Ashwell F Bornet E B FernandM B Roberfroid ldquoScientific concepts of functional foods in

Journal of Chemistry 11

Gln

NR NiR

Glu Glu

Protein

Gln 2OG

NR NiRGS

TCA

TSNAs

Mo

NR Protein

NR GS

In fastgrowthperiod

At maturitystage

Leaf

Leaf

Nitratestorage

Nitrateaccumulation

Leaf

Nitratetransport

Glu Glu

Leaf

2OG

N（3

N（3

N（3

N3-N

N3-N

N3-N

N2-N

N2-N

Nx + lkaloid

MＩ + fo

Figure 8 Mechanisms for decreasing nitrate and TSNA concentrations in tobacco by spraying regulating chemicals Gfo glufosinate NRnitrate reductase NRA nitrate reductase activity NiR nitrite reductase GS glutamine synthetase GSA glutamine synthetase activity Glnglutamine Glu glutamate and OG oxaloacetate After sprayingMo on tobacco during the fast-growth period nitrate significantly decreasedwhile NRA and soluble protein content increasedThese decreased the amount of nitrate storage and promoted tobacco development duringthe fast-growth period After spraying Mo during the fast-growth stage and spraying Mo and Gfo at the stage of maturity NRA increasedand GSA decreased in tobacco which can significantly reduce nitrate accumulation and TSNA formation by nitrogen loss due to ammoniavolatilization

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank Editage (httpswwweditagecom) forEnglish language editing and publication support

References

[1] H R Burton N K Dye and L P Bush ldquoRelationship betweentobacco-specific nitrosamines and nitrite from different air-cured tobacco varietiesrdquo Journal of Agricultural and FoodChemistry vol 42 no 9 pp 2007ndash2011 1994

[2] B Siminszky L Gavilano S W Bowen and R E DeweyldquoConversion of nicotine to nornicotine in Nicotiana tabacumis mediated by CYP82E4 a cytochrome P450 monooxygenaserdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 41 pp 14919ndash14924 2005

[3] IARC ldquoSmokeless tobacco and some tobacco-specific N-nitrosamines monographs on the evaluation of carcinogenicrisks to humansrdquo National Publishing Group vol 89 Article ID1e592 2005

[4] H Shi R Wang and L P Bush ldquoThe relationships betweenTSNAs and their precursors in burley tobacco from differentregions and varieties JournalofFoodrdquoAgricultureEnvironmentvol 10 no 2 pp 1048ndash1052 2012

[5] H R Burton G H Childs Jr R A Andersen and P D Flem-ing ldquoChanges in chemical composition of Burley tobacco dur-ing senescence and curing 3 Tobacco-specific nitrosaminesrdquoJournal of Agricultural and Food Chemistry vol 37 no 2 pp426ndash430 1989

[6] H Shi R Wang L P Bush et al ldquoChanges in TSNA Contentsduring Tobacco Storage and the Effect of Temperature andNitrate Level on TSNA Formationrdquo Journal of Agricultural andFood Chemistry vol 61 pp 11588ndash11594 2013

[7] K Fytianos and P Zarogiannis ldquoNitrate and nitrite accumula-tion in fresh vegetables from Greecerdquo Bulletin of EnvironmentalContamination and Toxicology vol 62 no 2 pp 187ndash192 1999

[8] Y Y Wang P K Hsu and Y F Tsay ldquoUptake allocation andsignaling of nitraterdquo Trends in Plant Science vol 17 no 8 pp458ndash467 2012

[9] K S Reddy and R C Menary ldquoNitrate reductase and nitrateaccumulation in relation to nitrate toxicity in Boronia meg-astigmardquo Physiologia Plantarum vol 78 no 3 pp 430ndash4341990

[10] Y Han Q Liao Y Yu et al ldquoNitrate reutilization mechanismsin the tonoplast of two Brassica napus genotypes with differentnitrogen use efficiencyrdquoActa Physiologiae Plantarum vol 37 no2 2015

[11] H Kosaka K Imaizumi K Imai and I Tyuma ldquoStoichiometryof the reaction of oxyhemoglobin with nitriterdquo BBA - ProteinStructure vol 581 no 1 pp 184ndash188 1979

[12] P Santamaria ldquoNitrate in vegetables toxicity content intakeand EC regulationrdquo Journal of the Science of Food and Agricul-ture vol 86 no 1 pp 10ndash17 2006

[13] A T Diplock P J Aggett M Ashwell F Bornet E B FernandM B Roberfroid ldquoScientific concepts of functional foods in

12 Journal of Chemistry

europe consensus documentrdquo British Journal of Nutrition vol81 supplement 1 pp 1ndash28 1999

[14] CM Onyango J Harbinson J K Imungi S S Shibairo andOvan Kooten ldquoInfluence of organic and mineral fertilization ongermination leaf nitrogen nitrate accumulation and yield ofvegetable amaranthrdquo Journal of Plant Nutrition vol 35 no 3pp 342ndash365 2012

[15] I S Vieira E P Vasconcelos andA AMonteiro ldquoNitrate accu-mulation yield and leaf quality of turnip greens in response tonitrogen fertilisationrdquo Nutrient Cycling in Agroecosystems vol51 no 3 pp 249ndash258 1998

[16] K Reinink and A H Eenink ldquoGenotypical differences innitrate accumulation in shoots and roots of lettucerdquo ScientiaHorticulturae vol 37 no 1-2 pp 13ndash24 1988

[17] I G Burns K Zhang M K Turner et al ldquoScreening forgenotype and environment effects on nitrate accumulation in24 species of young lettucerdquo Journal of the Science of Food andAgriculture vol 91 no 3 pp 553ndash562 2011

[18] F C Olday A V Barker and D N Maynard ldquoA physiologicalbasis for different patterns of nitrate accumulation in twospinach cultivarsrdquo JournaloftheAmericanSocietyofHorticultur-alScientists vol 101 pp 217ndash219 1976

[19] Y Tang X Sun C Hu Q Tan and X Zhao ldquoGenotypic dif-ferences in nitrate uptake translocation and assimilation of twoChinese cabbage cultivars [Brassica campestris L ssp Chinen-sis(L)]rdquo Plant Physiology and Biochemistry vol 70 pp 14ndash202013

[20] R R Mendel and F Bittner ldquoCell biology of molybdenumrdquoBiochimica et Biophysica Acta (BBA) - Molecular Cell Researchvol 1763 no 7 pp 621ndash635 2006

[21] M Farooq A Wahid and K H M Siddique ldquoMicronutrientapplication through seed treatmentsa reviewrdquo Journal of SoilScience and Plant Nutrition vol 12 no 1 pp 125ndash142 2012

[22] C M J Williams N A Maier and L Bartlett ldquoEffect ofmolybdenum foliar sprays on yield berry size seed formationand petiolar nutrient composition of ldquoMerlotrdquo grapevinesrdquoJournal of Plant Nutrition vol 27 no 11 pp 1891ndash1916 2004

[23] G A Biscaro S A R Goulart Jr and R P Soratto ldquoMolybde-num applied to seeds and side dressing nitrogen on irrigatedcommon bean in cerrado soilrdquoCiΩnciaaAgrotecnologia vol 33pp 1280ndash1287 2009

[24] K Ramesh and V Thirumurugan ldquoEffect of seed pelleting andfoliar nutrition on growth of soybeanrdquo Madras AgriculturalJournal vol 88 pp 465ndash468 2001

[25] R D Blackwell A J S Murray and P J Lea ldquoInhibition ofphotosynthesis in barley with decreased levels of chloroplasticglutamine synthetase activityrdquo Journal of Experimental Botanyvol 38 no 11 pp 1799ndash1809 1987

[26] A Wild H Sauer and W Ruhle ldquoThe effect of phos-phinothricin (glufosinate) on photosynthesis I Inhibition ofphotosynthesis and accumulation of ammoniardquo Zeitschrift furNaturforschung - Section C Journal of Biosciences vol 42 no 3pp 263ndash269 1987

[27] H Sauer A Wild and W Ruhle ldquoThe effect of phos-phinothricin (glufosinate) on photosynthesis ii the causes ofinhibition of photosynthesisrdquo Zeitschrift fur Naturforschung -Section C Journal of Biosciences vol 42 no 3 pp 270ndash278 1987

[28] A Wild and C Wendler ldquoEffect of glufosinate (phos-phinothricin) on amino acid content photorespiration andphotosynthesisrdquo PesticideScience vol 30 pp 422ndash424 1991

[29] C Wendler M Barniske and A Wild ldquoEffect of phos-phinothricin (glufosinate) on photosynthesis and photorespira-tion of C3 and C4 plantsrdquo Photosynthesis Research vol 24 no 1pp 55ndash61 1990

[30] J F Seelye W M Borst G A King P J Hannan and DMaddocks ldquoGlutamine synthetase activity ammonium accu-mulation and growth of callus cultures of Asparagus officinalisL exposed to high ammonium or phosphinothricinrdquo Journal ofPlant Physiology vol 146 no 5-6 pp 686ndash692 1995

[31] R Manderscheid S Schaaf M Mattsson and J K SchjoerringldquoGlufosinate treatment of weeds results in ammonia emissionby plantsrdquo Agriculture Ecosystems amp Environment vol 109 no1-2 pp 129ndash140 2005

[32] Q Zhang Q Song C Wang C Zhou C Lu and M ZhaoldquoEffects of glufosinate on the growth of and microcystin pro-duction by Microcystis aeruginosa at environmentally relevantconcentrationsrdquo Science of the Total Environment vol 575 pp513ndash518 2017

[33] D Xu J Sun and H Yang ldquoInhibitory effects of enzymes onnitrogen metabolism at mature stage and quality of curedtobacco leavesrdquo JournalofTobaccoScienceTechnology vol 49 no3 pp 17ndash23 2016

[34] H Zhang X Liu and J Zhang ldquoMechanism and utilizationof glufosinate-ammoniumrdquo PesticideScienceandAdministrationvol 25 no 4 pp 23ndash27 2005

[35] B A Sellers R J Smeda and J Li ldquoGlutamine synthetaseactivity and ammonium accumulation is influenced by time ofglufosinate applicationrdquo Pesticide Biochemistry and Physiologyvol 78 no 1 pp 9ndash20 2004

[36] N KMoustakas andHNtzanis ldquoDrymatter accumulation andnutrient uptake in flue-cured tobacco (Nicotiana tabacum L)rdquoField Crops Research vol 94 no 1 pp 1ndash13 2005

[37] National Tobacco Institute of Greece Guidelines for TobaccoProduction 1996

[38] S D Bao Agricultural and Chemistry Analysis of Soil Agricul-ture Press Beijing (in Chinese 2005

[39] H S Li Principle and technology of plant physiological andbiochemical experiments Higher EducationPress Beijing 2000

[40] D OrsquoNeal and K W Joy ldquoGlutamine synthetase of pea leavesrdquoJournal of Plant Physiology vol 54 no 5 pp 773ndash779 1974

[41] F J Dentener and P J Crutzen ldquoA three-dimensional model ofthe global ammonia cyclerdquo Journal of Atmospheric Chemistryvol 19 no 4 pp 331ndash369 1994

[42] J D Crutchfield and J H Grove ldquoA new cadmium reductiondevice for the microplate determination of nitrate in water soilplant tissue and physiological fluidsrdquo Journal of AOAC Interna-tional vol 94 no 6 pp 1896ndash1905 2011

[43] A Jack and L Bush ldquoThe ldquoLCrdquo protocol ndash Appendix 3Laboratory Proceduresrdquo pp 21-23 University of KentuckyLexington USA (2007)rdquo httpwwwukyeduAgTobaccoPdf327LC-Protocol

[44] X Wei X Deng D Cai et al ldquoDecreased tobacco-specificnitrosamines by microbial treatment with Bacillus amylolique-faciens DA9 during the air-curing process of burley tobaccordquoJournal of Agricultural and Food Chemistry vol 62 no 52 pp12701ndash12706 2014

[45] W Morgan J Reece C Risner et al ldquoA collaborative study forthe determination of tobacco specific nitrosamines in tobaccordquoBeitrage zur Tabakforschung International vol 21 no 3 pp 192ndash203 2014

Journal of Chemistry 13

[46] J Zhou R Bai and Y Zhu ldquoDetermination of four tobacco-specific nitrosamines in mainstream cigarette smoke by gaschromatographyion trap mass spectrometryrdquo Rapid Commu-nications in Mass Spectrometry vol 21 no 24 pp 4086ndash40922007

[47] SY Ai J W Yao and X H Huang ldquoStudy on the nitratereduction characteristic of vegetablesrdquo Plant Nutrition andFertilizer Science vol 8 no 1 pp 40ndash43 2002

[48] Y Li D Chang J Sun H Yang JWang andH Shi ldquoDifferenceof nitrogen metabolism between flue-cured tobacco and burleytobacco seedlingsrdquo Tobacco Science amp Technology vol 50 no 1pp 6ndash11 2017

[49] Z Q Shang ldquoEffects of nitrogen amount on growth anddevelopment yield and quality in burley tobaccordquo ChineseAgricultural Science Bulletin vol 23 pp 299ndash301 2007

[50] Z H Li Z Song andGHuang ldquoEffects ofmolybdenum apply-ing in tobacco field on photosynthesis nitrogen metabolismand quality of tobaccordquo Tobacco Science amp Technology vol 11pp 56ndash58 2008

[51] N Nautiyal and C Chatterjee ldquoMolybdenum Stress-InducedChanges in Growth and Yield of Chickpeardquo Journal of PlantNutrition vol 27 no 1 pp 173ndash181 2004

[52] N C Gopalachari A Sastry and D Rao ldquoEffect of maturityof leaf at harvest on some physical and chemical properties ofcured leaf of Delcrest flue-cured tobacco NicotianatabacumLrdquo IndianJournalofAgriculturalScience vol 10 pp 901ndash911 1970

[53] Z Zhang S Xiong Y Wei X Meng X Wang and X Ma ldquoTherole of glutamine synthetase isozymes in enhancing nitrogenuse efficiency ofN-efficientwinterwheatrdquo Scientific Reports vol7 no 1 2017

[54] J Wang H Yang H Shi et al ldquoNitrate and Nitrite Pro-mote Formation of Tobacco-SpecificNitrosamines viaNitrogenOxides Intermediates during Postcured Storage under WarmTemperaturerdquo Journal of Chemistry vol 2017 pp 1ndash11 2017

[55] S Liang Study on mechanism of exogenous substances reducingTSNA content in burley tobacco [Master thesis] Masters Disser-tation Wuhan Huazhong Agriculture University 2013

[56] H Liu J Han and S Yang ldquoEffect of malonate acid on nicotinein burley tobaccordquo ActaTobacariaSinica vol 6 no 3 pp 47-482000

[57] R S Lewis R G Parker D A Danehower et al ldquoImpact ofalleles at the Yellow Burley (Yb) loci and nitrogen fertilizationrate on nitrogen utilization efficiency and tobacco-specificnitrosamine (TSNA) formation in air-cured tobaccordquo Journal ofAgricultural and Food Chemistry vol 60 no 25 pp 6454ndash64612012

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 201

International Journal ofInternational Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal ofInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of