Research Article Functional Characterization of 9-/13-LOXs ...downloads.hindawi.com/journals/bmri/2016/4275904.pdf · Here, RNA interference- (RNAi-) induced gene expression inhibition

Research ArticleFunctional Characterization of 9-13-LOXs in Rice andSilencing Their Expressions to Improve Grain Qualities

Moytri RoyChowdhury1 Xiaobai Li23 Hangying Qi4 Wenxu Li5

Jian Sun2 Cheng Huang4 and Dianxing Wu2

1Department of Horticulture Washington State University Pullman WA 99164 USA2State Key Lab of Rice Biology International Atomic Energy Agency Collaborating Center Zhejiang UniversityHangzhou Zhejiang 310029 China3Zhejiang Academy of Agricultural Sciences Hangzhou Zhejiang 310021 China4Agricultural Extension Extending Stations Shaoxing amp Zhuji Agricultural Bureau Shaoxing Zhejiang 312000 China5Institute for Wheat Research Henan Academy of Agricultural Sciences Zhengzhou Henan 450002 China

Correspondence should be addressed to Xiaobai Li hufanfan1982815gmailcom and Dianxing Wu dxwuzjueducn

Received 5 December 2015 Accepted 24 April 2016

Academic Editor Sudhir Sopory

Copyright copy 2016 Moytri RoyChowdhury et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Lipoxygenases (LOXs) are involved in oxidative rancidity and render rice unsuitable for human consumption Here RNAinterference- (RNAi-) induced gene expression inhibition was used to analyze the functions of the branseed-specific LOXs inrice r9-LOX1 and L-2 (9-LOX category) were the candidate genes expressing a branseed-specific LOX while RCI-1 was (13-LOXcategory) a plastid-specific LOX Real-time PCR showed that three LOXs were cultivartissue specific expression on a certain levelr9-LOX1 and L-2were generally much higher in active branseed than in stabilized bran mature seed and regenerated plant RCI-1was barely expressed in seed In transgenic lines r9-LOX1 as well as L-2 expression was dramatically downregulated compared tothe nontransgenic controls SPMEGC-MS analysis of r9-LOX1 RNAi transgenic lines showed 7433 decrease in nonanal content(formed during oxidation of linoleic acid by lipoxygenase) but 38824 increase in acetic acid and 18484hexanal (direct productsof 13-LOX) These results indicate that r9-LOX1 positively regulates the amount of nonanal but negatively regulates acetic acid andhexanal The negative regulation may be due to a mechanism of negative feedback between LOX family membersThe informationwill help comprehensively understand the function of the branseed-specific LOXs r9-LOX1 and improve the storage quality inthe future

1 Introduction

Lipoxygenases (LOXs) ubiquitously exist in the seeds ofmanyplant species Seed LOXs may be involved in the fatty acidperoxidation lipids storage production of growth regulatorsresponses to pathogens and nitrogen storage [1] The influ-ence of LOXs on favor quality is reported in soybean and riceSoybean LOXs are involved in the production of volatile com-pounds (such as n-hexanal) which are associated with grassybeany and rancid off-flavors in soybean and soy foods [2]In stored rice seeds LOX activities are also present [3] and

hexanal is the predominant component of stale off-flavorsderived from lipid peroxidation [4] Rice seed LOXs are alsoinvolved in lipid peroxidation in rice seeds [5] Since theabsence of LOXs can alleviate seed lipid peroxidation LOXsare always the targets of genetic modifying during molecularbreeding Soybean ldquoIA2025rdquo a LOX triple null-mutationsoybean line can be used to produce tofu and soymilkwith improved flavor and sensory qualities [6ndash8] In barleya LOX-1 deficient line ldquoDaikeiLM1rdquo is gained from ldquoKarlM2rdquo through sodium azide treatment [9] This mutated linecan effectively improve flavor stability in beer [10] In rice

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 4275904 8 pageshttpdxdoiorg10115520164275904

2 BioMed Research International

Table 1 Primer sequences of three LOX genes for cloning

Gene (accession number) Primers Sequence Product size

r9-LOX (AB099850) CDS r9-LOX1F CACCGCTGGACGAGAACCCAGAGAAG 238 bpCDS r9-LOX 1R CAGGGGGTCCTTGTTCATGTT

RCI-1 (AJ270938) CDSRCI-1F CACCCGAAGGCTACTTCAGGGAGGTG 214 bpCDSRCI-1R CGTGTCCCTGACAAGTTGGATG

L-2 (X64396) CDSL-2F CACCCCTCGCCATCTACTACCCCAACG 217 bpCDSL-2R GTACGGGTACTGCCCGAAGTTG

the absence of sLOX3 in the seeds of ldquoDawDamrdquo results inless stale flavor [11]

According to the differences of substrates at carbon 9 or13 of the hydrocarbon backbone LOXs are classified into 9-LOXs and 13-LOXs [12] Some plants may have predominant9-LOX or 13-LOX activity while some other plants mayhave equally 9-LOX and 13-LOX activities According to theprotein sequences LOXs are classified into three types asdescribed by Mizuno et al [13] Type I is localized in chloro-plast and stress inducible Type II is localized in cytoplasmderived fromdicots and not stress inducible Type III is local-ized in the cytoplasm derived from monocots and relatedto seed germination Type I LOXs have a transit peptidelacking in Type II and Type III LOXsThree isozymes in TypeIII LOXs (LOX1L-1 LOX2L-2 and LOX3L-3) have beenidentified inOryza sativa embryos Among them LOX3 is themost abundant one [14] The catalytic LOX domains in bothType II and Type III LOXs have oxygen binding and oxylipinsynthesis sites The enzyme r9-LOX1 belongs to Type III andis the focus of this study Three Type III LOXs (L-1 L-2 andL-3) in developing rice seeds have been identified [15] L-2(Type III) derives from 3-day-old seedlings [16] while RLL(Type I) is isolated from rice leaves [17] L-3 (Type III) is themajor component of these isozymes and accounts for 80ndash90 of their total activities [18] The lack of L-3 in DawDamleads to a decrease of lipid peroxidation and the alleviationof stale flavor accumulation by hexanal as well as pentanaland pentanol compounds during storage [19] UnfortunatelyDawDam could not be used for rice production due to itspoor agronomic characters [18] It is speculated that the lackof LOX-3 may affect the metabolism in the whole plant

In order to effectively improve rice grain storage qualitiesinactivation of some lipase andor LOXs should be achievedwithout compromising nutritional value and agronomicqualities RNAi could be an ideal method to achieve this goalbased on its applications on improving the nutritional qualityof various crops [20] Therefore the objectives of this studywere to select two candidate seedbran LOXs gene(s) developsuitable constructs for stable RNAi in rice and study thepotential benefits of silencing branseed-specific LOXs

2 Materials and Methods

21 Plant Materials The japonica rice cultivar ldquoLaGruerdquo wasused to clone target genes and develop transgenic plants Thetissues from DawDam Taipei-309 and transgenic lines wereused for gene expression analysis

22 Genomic DNA Extraction and PCR Analysis GenomicDNA was extracted from the leaf tissues of Taipei-309 seed-lings byNucleonPhytopure PlantDNAExtraction kit (Amer-sham Biosciences) according to the manufacturersrsquo protocol

Two Type III LOXs r9-LOX1 (accession numberAB099850) and L-2 (accession number X64396) and a leaf-specific LOX RCI-1 (accession number AJ270938) as well asRCI-1 (accession number AJ270938) were selected for studyInterestingly the L-3 (accession number E03480) sequencewas noted to be identical to the L-2 sequence (accessionnumber X64396) known to be distributed in rice embryo andhave C9 specific LOX activity [13] The nucleotide sequencesof L-2 and L-3 (Type III) were identicalThe primers for theseLOX genes were listed in Table 1 Gene sequences for invertedrepeats were amplified using thermostable proofreadingDNA polymerase from Stratagene (La Jolla CA USA) TheCACC sequence was added to the 51015840 end of forward primerfor facilitating directional incorporation into InvitrogenrsquospENTRD-TOPO entry vector PCR was performed by usingKOD-Plus-Neo (TOYOBO Japan) and then the productswere separated by 07 agarose gelThe target fragments werecollected using Qiangen gel purification kit

23 Construction of hpRNAi Vectors RNAi constructs wereprepared following the Gateway System protocol [21] Forconstructing hpRNAi vectors the target gene fragments werefirstly cloned into TOPO pENTR following the GatewaySystem protocol (Invitrogen Carlsbad CA)The transforma-tion of Agrobacterium tumefaciens EHA105 with the pANDAvector was performed using the Freeze and Thaw method[22] Agrobacterium-mediated transformation in rice wascarried out as described previously [23 24] withminormod-ifications [25]The positive control EHA105 was transformedwith pWHNG vector

24 Rice Transformation The transformation of Agrobac-terium tumefaciens EHA105 with the pANDA vector wasperformed using the Freeze andThawmethod [22] Agrobac-terium-mediated transformation in rice was carried out asdescribed previously [23 24] with minor modifications [25]The positive control EHA105 was transformed with pWHNGvector

Transformation was carried out using 21-day-old calligrown on B5 medium with 22mgL 2 4D After beingtreated with liquid cocultivation medium (YEP containingEHA105 at 600OD of 1) for 20min the calli were transferredto a solid cocultivation medium (B5 medium with 22mgL

BioMed Research International 3

Table 2 Rate of Agrobacterium-mediated transformation

Constructs Total explants Transformed plants Regenerationpercentage () Confirmed transformation Transformed plants

percentage ()Negative control 7 4 570EHA105 + pWHNG 40 4 100 2 50EHA105 + r9-LOX 160 12 75 4 20EHA105 + L2 200 22 110 7 30EHA105 + RCI-1 180 12 66 5 30Mean 878 325

2 4D and 100 um acetosyringone) for 3 days After cocultiva-tion the transformed calli were cultured in solid B5 mediumwith 22mgL 2 4D and 250mgL Claforan for 7 days andthen transferred to the selective medium (B5 medium with22mgL 2 4D 250mgL Claforan and 25mgL geneticin)for 15 days The proliferated yellowish white calli weretransferred to the samemedium for another 15 days Resistantcalli were transferred to regeneration medium (B5 mediumwith 003mgL Picloram and 035mgL BA) for 15 days After15 days the medium was replaced with fresh medium foranother 15 days Seedlings were then transferred to fresh B5medium without antibiotics for 15 days and then transferredto the planters in greenhouse

25 Real-Time PCR Total RNA was extracted from matureseed 2-day-old germinating seed imbibed seed and stabi-lized bran and 35-day-old regeneration plants of differentvarieties rice or transgenic plants using Trizol (InvitrogenCarlsbad CA) [26] and treated with RNase-free DNase I(TaKaRa Shuzo Kyoto Japan) to remove potential contam-inating genomic DNA First Strand cDNA was synthesizedusing SuperScript TM II RT kit (Invitrogen Carlsbad CA)

All samples were amplified in triplicate from the sameRNA preparation and the mean values were calculated Theprimers for real-time analysis were designed based on genespecific sequences (see Supplementary Table S1 in Supple-mentaryMaterial available online at httpdxdoiorg10115520164275904) RT-PCR was performed by using SyberGreenreg (Roche Diagnostics) on C1000trade Thermal Cyclerequipped with CFX96trade Real-Time System (Bio-Rad USA)Primer specificitywas tested by isolated PCRproducts in highresolution gel electrophoresis Melting curve program (60ndash95∘C) was performed with the heating rate at 010∘Csec con-tinuous fluorescencemeasurement and a final cooling step to40∘C

To determine relative amount of LOXs mRNA transcriptin different rice tissues we applied the REST (RelativeExpression Software Tool) calculations based on the Ct (cyclethreshold) values at a constant fluorescence level [27 28]Therelative expressionmRNA transcript ratio of each LOX wascomputed based on its RT-PCR efficiency and the crossingpoint difference between LOX and Actin control

26 SPME GCMS Analysis Samples for SPMEGCMS wereprepared by first hulling the seeds using hands or table toprice huller depending on the amount of rice and the easiness

to hull the seeds The hulled seeds were then milled to 10(removal of bran layer) degree using ldquoWonderHand Millrdquo[29] The native and transgenic bran was subjected to iden-tical storage conditions to maintain identical moisture levelSPME GCMS analysis was performed using a methodmodified by Grimm et al [30]

3 Results

31 RNAi Vector Construct and Development of TransgenicPlants PCR results showed the correct sizes of LOX frag-ments with the range of 200ndash300 bp for 9r-LOX1 and L-2(Supplementary Figure S1)The fragments of LOX genes weretargets for RNAi shown as Figure 1 These fragments wereinitially linked to TOPO pENTR vector and then transferredinto pANDA for hpRNAi expression (Supplementary FigureS2)The insertions of target fragments in pENTER vector andin pANDA were confirmed by PCR and restriction enzymedigestion (Supplementary Figures S3 and S4) Sequencingresults revealed that all the sequences for r9-LOX1 L-2 andRCI-1 were consistent with the original sequences in thedatabase only with a few mismatches Moreover the binarypWHNG vector containing GUS was chosen as positive con-trol ofAgrobacterium-mediated transformation (Supplemen-tary Figure S5)The hpRNAi vector and pWHNGwere trans-formed into rice by Agrobacterium-mediated transformationof rice mature embryo A total of 160 to 200 explants wereused for r9-LOX1LOX2 orRCI-1 gene silencing plants and 40explants were for generation of ldquopositive controlrdquo transgenicplants After selection and regeneration 12 to 22 transformedT0plants were produced for hpRNAi vectors and four for

pWHNG vector with average regeneration rate of 878Thetransgenic plantswere further confirmedbyPCRusing vectorspecific primers (Supplementary Figure 6 and Table 2) Theresult showed that two to seven PCR T

0plants were positive

for the vectors with average rate of positive confirmation of325 The plants were grown in greenhouse until matureand then T

1seeds were harvested and grown for two gener-

ations to obtain stable transgenic plants The different tissueswere collected for the expression analysis of target geneSchematic diagram for the transformation process is depictedin Figure 2

32 Expression Pattern of LOXs This study analyzed theexpression patterns of two branseed-specific genes r9-LOX1 and L-2 and one chloroplast-specific gene RCI-1


r9-LOX1 (accession number AB099850)ATG TGA

RCI-1 (accession number AJ270938)ATG TGA

CDS RCI-1F CDS RCI-1R

L-2 (accession number X64396)ATG TGA

CDS L-2F CDS L-2R

CDSr9-LOX1F CDSr9-LOX1R

2900bp

Figure 1 Gene structure and the target site for RNA interference

Embryo scutellum-derived callus (a) Embryogenic nodular units (b arrows)

GUS activity at the surface of a cocultured

callus (d)

Callus transferred to fresh medium to reach an optimal size (c)

Transgenic plants

Transgenic plants in green house

Induction of primary callus

Transfer to fresh regeneration medium

Transfer to plantregeneration medium

Transfer to fresh selection medium

Mature seed

21 days

7 daysLiquid cocultivation

medium

3 days

Blot dried and transferred to solid cocultivation medium

Transfer to medium with positive selection for transformed cells

15 days

15 days15 days15 days

Transgenic plants regenerating from callus

15 days

Selected calli ready for regeneration

Calli after first transfer toselective medium

Maturation ofregenerated plantlets

Plants grown to maturity

EHA 105 in YEP at OD 600 of 1

Transfer to medium with antibiotics to suppress Agrobacterium

B5-0

(a) (b)

(c)

(d)

(e)(f)(g)

(h)

(i)

B5 + 22mgL 2 4-D +250mgL Claforan

B5 + 22mgL 2 4-D +250mgL Claforan +25mgL geneticin

035mgL BAB5 + 003mgL Pic +

20minsB5 + 22mgL 2 4-D

100120583m ASB5 + 22mgL 2 4-D +

Figure 2 Agrobacterium-mediated transformation The figure is a schematic diagram of the events involved in rice transformation

(Figures 3(a) 3(b) and 3(c)) r9-LOX1 was highly expressedin germinating and imbibed seed as well as Spring and Taipeibran L-2 was also highly expressed both in germinating andimbibed seed as well as Taipei bran In contrast r9-LOX1 andL-2 were both barely expressed in transgenic tissues RCI-1

was highly expressed in ldquonullrdquo mutant ldquoDawDamrdquo imbibedseed as well as transgenic tissue

The expressions of these three LOXs showed tissuecultivar-specificity on a certain level (Figures 3(a) 3(b) and3(c)) r9-LOX1 expressionwas generallymuchhigher in active


0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 A10 A11 A12

Relat

ive e

xpre

ssio

n le

vel

(1) Mature seed(2) Germinating seed(3) Imbibed seed(4) Stabilized bran(5) DawDam imbibed seed(6) Regenerated plant

(7) DawDran bran(8) Spring bran(9) Taipei bran(A10) Transgenic r9-LOX1 callus(A11) Transgenic r9-LOX1 plant(A12) Transgenic r9-LOX1 seed

Plant tissue

(a)

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 B10 B11 B12

Relat

ive e

xpre

ssio

n le

vel


(7) DawDran bran(8) Spring bran(9) Taipei bran(B10) Transgenic L-2 callus(B11) Transgenic L-2 plant(B12) Transgenic L-2 seed

Plant tissue

(b)

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 C10

Relat

ive e

xpre

ssio

n le

vel

C11 C12


(7) DawDran bran(8) Spring bran(9) Taipei bran(C10) Transgenic RCI-1 callus(C11) Transgenic RCI-1 plant(C12) Transgenic RCI-1 seed

Plant tissue

(c)

Figure 3 Relative expressions of three LOX genes (a) r9-LOX1 (b) L-2 and (c) RCI-1 among transgenic and nontransgenic rice tissues

branseed than in stabilized bran mature seed and regen-erated plant Among active branseed tissue germinatingseeds had the highest expression of r9-LOX1 followed byimbibed seed Taipei bran and Spring bran Similarly L-2had relative high expression in active branseed tissue Inter-estingly the chloroplast-specific RCI-1 showed exceedinglyhigh expression in the imbibed seeds of the null mutantsldquoDawDamrdquo and in all of the transgenic calluses plants andseeds (Figure 3(c))

33 The Volatile Byproducts in r9-LOX1 RNAi Rice Bran Thevolatile byproducts during lipid oxidation in nontransgenicand transgenic RNAi rice bran were examined by SPMEGC-MS Acetic acid and hexanal are the direct products of 13-lipoxygenase while nonanal is formed during the oxidationof linoleic acid by LOX In SPMEGC-MS analysis silencingr9-LOX1 coding region was obviously associated with the

changes of byproducts during lipid oxidation The contentsof acetic acid were much higher in transgenic bran than inthe control lines by 38824 as well as hexanal by 18484 Incontrast the contents of nonanal were significantly decreasedin transgenic lines by 7433 (Figure 4)

4 Discussion

41 RNAi Approach for Nutrition Improvement and GeneticStudy in Crops HGS-hpRNAi has been used to improvenutritional qualities in several crops For example it is usedto successfully reduce 120574-Gliadins in breadwheat [31] Besidesit is used to reduce the expression levels of two starch-branching enzymes SBEIIa and SBEIIb which results inincreased amylose content [32] In transgenic peanut knock-ing down oleate desaturase (FAD2) by RNAi can increaseoleic acid content by 70 [33] Moreover the application of


Products in pathway

Acetic acid

Hexanal

Nonanal

Naphthalene

GC RT (mins)

244

575

1511

1773

Control area counts

6866451

9019178

22363505

117817908

Transgenic area counts

33524756

25689932

5738826

0

Note r9-LOX1 RNAi lines showed 7433 decrease in nonanal but 38824 increase in acetic acid and 18484 hexanal

Abun

danc

e

400 600 800 1000 1200 1400 1600 1800ControlSample

Naphthalene

NonanalHexanal

Hexanol

Acetic acid

Time

14e + 0712e + 071e + 078e + 066e + 064e + 062e + 06

0

lowastlowast

lowast

lowast

lowastA SPME contaminant

Figure 4 SPMEGC-MS analysis of the volatile byproducts by lipid oxidation in nontransgenic and transgenic rice bran of r9-LOX1

this technology in our research could provide more insightsinto the functional characterization of rancidity-associatedLOXs Traditionally studies of gene functions mainly reliedon traditional breeding strategies which are time consumingIn the recent decade RNAi have largely expedited thisprocess In this study pANDA vector was adopted since itis an efficient RNAi vector for rice and has been used forAgrobacterium transformation in rice over the years [21] Ourresults showed that two branseed-specific LOXsrsquo expressionwas suppressed in more than 90 of the transgenic plantsindicating the high efficiency of RNAi system

42 LOXsrsquo Expression Patterns The expression of two branseed-specific LOXs and one plastid-specific LOXwere studiedamong different tissues Two branseed-specific genes r9-LOX1 and L-2 were both highly expressed in active seedincluding imbibed and germinating seed but barely expressedin stabilized bran mature seed In previous studies it isreported that r9-LOX1 is expressed in rice imbibed seeds [13]and in tea plant seeds [34] The similar expression patternof the two branseed-specific LOXs may be due to theirsimilar biological functions in rice branseeds and 713homology [13] Compared to r9-LOX1 L-2 showed about15 decreased expression in germinating and imbibed seedsand less expression in Spring bran The results are consistentwith the previous study where an expressional variationis observed between the predicted branseed-specific LOXsin soybean [35] The different expression patterns of LOXisozymes indicate that theymay also play other different rolesduring seed developmental stages

The expression patterns of r9-LOX1 and L-2 were sig-nificantly different from that of RCI-1 (Figures 3(a) 3(b)and 3(c)) The expressions of the two branseed-specificLOXs were low in null-mutation imbibed seed and rare inthe transgenic T

1seeds with corresponding RNAi construct

(Figures 3(a) 3(b) and 3(c)) InterestinglyRCI-1 is previouslyreported to be chloroplast-specific [36] The chloroplast-specific RCI-1 showed an exceedingly high expression in the

imbibed seeds of the null mutants as well as an expression ata certain level in Taipei bran (Figure 3(c)) which indicatesthat we should take RCI-1 as plastid-specific rather than thepreviously reported leaf-specific In addition RCI-1 expres-sion was also observed in the seeds of transgenic lines (Fig-ure 3(c)) Compared to r9-LOX1 and L-2RCI-1 showed a highexpression level in the imbibed seeds of null-mutation lineDawDam whichmight be caused by amechanismof negativefeedback between Type III LOX members in seeds

43 The Biologic Functions of r9-LOX1 In SPMEGC-MSanalysis r9-LOX1 RNAi line showed 7433 reduction ofnonanal (formed during the oxidation of linoleic acid byLOX)These results indicate that r9-LOX1 positively regulatesthe amount of nonanal Previous study also discovers apositive correlation between nonanal concentration and LOXactivity [37] However r9-LOX1 RNAi lines showed 38824increase in the amount of acetic acid and 18484 increase inhexanal (direct products of 13-LOX) (Figure 3) whichmay bedue to a negative feedback mechanism between LOX familymembers Similarly RCI-1 RNAi line did not exhibit efficientsilencing effect whichmay be due to the shared oxylipin syn-thesis domain between Type III and Type II LOXs [38]Theseresults suggest that the LOXs derived from different cate-gories may coordinate with each other and play importantroles inmany biological processes For example the completeknockdown of Type III LOXs results in poor defense functionand therefore leads to poor agronomic traits in DawDam

5 Conclusion

The results suggest that r9-LOX1 plays roles in synthesis path-ways of nonanal acetic acid and hexanal in rice branseedNotably it is very hard to reduce all of LOX rancid byproductsby silencing LOXs because of the complementary action ofdifferent members of LOX family Since a number of LOXsalso play crucial parts in some basic biological processessilencing all of them in rice will result in plants with poor


agronomic characters like DawDam The information willhelp understand the function of LOXs and improve thestorage quality in the future

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Moytri RoyChowdhury Xiaobai Li and Hangying Qi equallycontributed to this paper

Acknowledgments

The authors would like to thank Dr Pace Lubinsky USDA-FAS and Junli Zhang University of California-Davis forcritical review of the paper USDA Dale Bumpers NationalRice Research Center for providing the rice seeds and DrCasey Grimm USDA-ARS for the assistance with GCMSexperiments The study was supported by Science Tech-nology Department of Zhejiang Province (0406) and theMajor State Basic Research Development Program of China(2013CBA01403)

References

[1] J Loiseau B L Vu M-H Macherel and Y Le DeunffldquoSeed lipoxygenases occurrence and functionsrdquo Seed ScienceResearch vol 11 no 3 pp 199ndash211 2001

[2] D S Robinson Z Wu C Domoney and R Casey ldquoLipoxyge-nases and the quality of foodsrdquo Food Chemistry vol 54 no 1pp 33ndash43 1995

[3] B S Shastry and M R R Rao ldquoStudies on lipoxygenase fromrice branrdquo Cereal Chemistry vol 52 pp 597ndash603 1975

[4] S Chikubu ldquoStale flavor of stored ricerdquo Jarq-Japan AgriculturalResearch Quarterly vol 5 pp 63ndash68 1970

[5] Q LongW Zhang PWang et al ldquoMolecular genetic character-ization of rice seed lipoxygenase 3 and assessment of its effectson seed longevityrdquo Journal of Plant Biology vol 56 no 4 pp232ndash242 2013

[6] A V Torres-Penaranda C A Reitmeier L A Wilson WR Fehr and J M Narvel ldquoSensory characteristics of soymilkand tofu made from lipoxygenase-free and normal soybeansrdquoJournal of Food Science vol 63 no 6 pp 1084ndash1087 1998

[7] D R Iassonova L A Johnson E G Hammond and S EBeattie ldquoEvidence of an enzymatic source of off flavors inlsquolipoxygenase- nullrsquo soybeansrdquo Journal of the American OilChemistsrsquo Society vol 86 no 1 pp 59ndash64 2009

[8] A V Torres-Penaranda and C A Reitmeier ldquoSensory descrip-tive analysis of soymilkrdquo Journal of Food Science vol 66 no 2pp 352ndash356 2001

[9] MOozeki T Nagamine TM Ikeda et al ldquoGenetic variation inlipoxygenase activity among Japanese malting barley cultivarsand identification of a new lipoxygenase-1 deficient mutantrdquoBreeding Research vol 9 no 2 pp 55ndash61 2007

[10] H Ye S Harasymow X-Q Zhang et al ldquoSequence variationand haplotypes of lipoxygenase gene LOX-1 in the Australianbarley varietiesrdquo BMC Genetics vol 15 article 36 2014

[11] Y Suzuki I Kazuo C Li I Honda Y Iwai and U MatsukuraldquoVolatile components in stored rice [Oryza sativa (L)] of

varieties with and without lipoxygenase-3 in seedsrdquo Journal ofAgricultural and Food Chemistry vol 47 no 3 pp 1119ndash11241999

[12] I Feussner and C Wasternack ldquoThe lipoxygenase pathwayrdquoAnnual Review of Plant Biology vol 53 pp 275ndash297 2002

[13] K Mizuno T Iida A Takano M Yokoyama and T FujimuraldquoA new 9-lipoxygenase cDNA fromdeveloping rice seedsrdquoPlantand Cell Physiology vol 44 no 11 pp 1168ndash1175 2003

[14] S Ida YMasaki and YMorita ldquoThe isolation ofmultiple formsand product specificity of rice lipoxygenaserdquo Agricultural andBiological Chemistry vol 47 no 3 pp 637ndash641 2014

[15] H Ohta S Ida B Mikami and Y Morita ldquoPurification andcharacterization of rice lipoxygenase component 3 from ricerdquoAgricultural and Biological Chemistry vol 50 pp 3165ndash31711986

[16] H Ohta Y Shirano K Tanaka Y Morita and D ShibataldquocDNA cloning of rice lipoxygenase L-2 and characterizationusing an active enzyme expressed from the cDNA inEscherichiacolirdquo European Journal of Biochemistry vol 206 no 2 pp 331ndash336 1992

[17] Y-L Peng Y Shirano H Ohta T Hibino K Tanaka and DShibata ldquoA novel lipoxygenase from rice Primary structure andspecific expression upon incompatible infection with rice blastfungusrdquo Journal of Biological Chemistry vol 269 no 5 pp 3755ndash3761 1994

[18] Z He Study on improving the storable character of rice grains byusing genetic engineering [PhD dissertation] Zhejiang Univer-sity 2004

[19] Y Suzuki andUMatsukura ldquoLipoxygenase activity inmaturingand germinating rice seeds with and without lipoxygenase-3 inmature seedsrdquo Plant Science vol 125 no 2 pp 119ndash126 1997

[20] G Tang G Galili and X Zhuang ldquoRNAi and microRNAbreakthrough technologies for the improvement of plant nutri-tional value and metabolic engineeringrdquo Metabolomics vol 3no 3 pp 357ndash369 2007

[21] DMiki andK Shimamoto ldquoSimple RNAi vectors for stable andtransient suppression of gene function in ricerdquo Plant and CellPhysiology vol 45 no 4 pp 490ndash495 2004

[22] S Y Dityatkin K V Lisovskaya N N Panzhava and B NIliashenko ldquoFrozen-thawed bacteria as recipients of isolatedcoliphage DNArdquo Biochimica et Biophysica Acta (BBA)mdashNucleicAcids and Protein Synthesis vol 281 no 3 pp 319ndash323 1972

[23] Y Hiei T Komari and T Kubo ldquoTransformation of rice medi-ated by Agrobacterium tumefaciensrdquo Plant Molecular Biologyvol 35 no 1-2 pp 205ndash218 1997

[24] S Toki ldquoRapid and efficient Agrobacterium-mediated transfor-mation in ricerdquo Plant Molecular Biology Reporter vol 15 no 1pp 16ndash21 1997

[25] M RoyChowdhury J A Dabul and P G C HubstenbergerldquoBiotechnology advances for Arkansas rice varieties (Oryzasativa L)rdquo Arkansas Academy of Science vol 60 pp 113ndash1182007

[26] P Chomczynski and N Sacchi ldquoSingle-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextractionrdquo Analytical Biochemistry vol 162 no 1 pp 156ndash1591987

[27] M W Pfaffl ldquoA new mathematical model for relative quantifi-cationin real-time RT-PCRrdquo Nucleic Acids Research vol 29 pp2002ndash2007 2001

[28] M W Pfaffl G W Horgan and L Dempfle ldquoRelative Expres-sion Software Tool (REST) for group-wise comparison and


statistical analysis of relative expression results in real-timePCRrdquo Nucleic Acids Research vol 30 no 9 article e36 2002

[29] R Bryant A Proctor M Hawkridge et al ldquoGenetic variationand association mapping of silica concentration in rice hullsusing a germplasm collectionrdquo Genetica vol 139 no 11-12 pp1383ndash1398 2011

[30] CCGrimmC Bergman J TDelgado andR Bryant ldquoScreen-ing for 2-acetyl-1-pyrroline in the headspace of rice usingSPMEGC-MSrdquo Journal of Agricultural and FoodChemistry vol49 no 1 pp 245ndash249 2001

[31] J Gil-Humanes F Piston A Hernando J B Alvarez P RShewry and F Barro ldquoSilencing of 120574-gliadins by RNA interfer-ence (RNAi) in bread wheatrdquo Journal of Cereal Science vol 48no 3 pp 565ndash568 2008

[32] A Regina A Bird D Topping et al ldquoHigh-amylose wheatgenerated by RNA interference improves indices of large-bowelhealth in ratsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 10 pp 3546ndash35512006

[33] D Yin S Deng K Zhan and D Cui ldquoHigh-oleic peanutoils produced by HpRNA-mediated gene silencing of oleatedesaturaserdquo Plant Molecular Biology Reporter vol 25 no 3-4pp 154ndash163 2007

[34] S Liu and B Han ldquoDifferential expression pattern of an acidic913-lipoxygenase in flower opening and senescence and in leafresponse to phloem feeders in the tea plantrdquo BMCPlant Biologyvol 10 article 228 pp 1471ndash2229 2010

[35] T K Park and J C Polacco ldquoDistinct lipoxygenase speciesappear in the hypocotylradicle of germinating Soybeanrdquo PlantPhysiology vol 90 no 1 pp 285ndash290 1989

[36] U Schaffrath F Zabbai and R Dudler ldquoCharacterizationof RCI-1 a chloroplastic rice lipoxygenase whose synthesisis induced by chemical plant resistance activatorsrdquo EuropeanJournal of Biochemistry vol 267 no 19 pp 5935ndash5942 2000

[37] Z Kong and D Zhao ldquoThe inhibiting effect of abscisic acidon fragrance of kam sweet ricerdquo Journal of Food and NutritionResearch vol 2 no 4 pp 148ndash154 2014

[38] S S Marla and V K Singh ldquoLOX genes in blast fungus (Mag-naporthe grisea) resistance in ricerdquo Functional and IntegrativeGenomics vol 12 no 2 pp 265ndash275 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Primer sequences of three LOX genes for cloning

Gene (accession number) Primers Sequence Product size

r9-LOX (AB099850) CDS r9-LOX1F CACCGCTGGACGAGAACCCAGAGAAG 238 bpCDS r9-LOX 1R CAGGGGGTCCTTGTTCATGTT

RCI-1 (AJ270938) CDSRCI-1F CACCCGAAGGCTACTTCAGGGAGGTG 214 bpCDSRCI-1R CGTGTCCCTGACAAGTTGGATG

L-2 (X64396) CDSL-2F CACCCCTCGCCATCTACTACCCCAACG 217 bpCDSL-2R GTACGGGTACTGCCCGAAGTTG

the absence of sLOX3 in the seeds of ldquoDawDamrdquo results inless stale flavor [11]

According to the differences of substrates at carbon 9 or13 of the hydrocarbon backbone LOXs are classified into 9-LOXs and 13-LOXs [12] Some plants may have predominant9-LOX or 13-LOX activity while some other plants mayhave equally 9-LOX and 13-LOX activities According to theprotein sequences LOXs are classified into three types asdescribed by Mizuno et al [13] Type I is localized in chloro-plast and stress inducible Type II is localized in cytoplasmderived fromdicots and not stress inducible Type III is local-ized in the cytoplasm derived from monocots and relatedto seed germination Type I LOXs have a transit peptidelacking in Type II and Type III LOXsThree isozymes in TypeIII LOXs (LOX1L-1 LOX2L-2 and LOX3L-3) have beenidentified inOryza sativa embryos Among them LOX3 is themost abundant one [14] The catalytic LOX domains in bothType II and Type III LOXs have oxygen binding and oxylipinsynthesis sites The enzyme r9-LOX1 belongs to Type III andis the focus of this study Three Type III LOXs (L-1 L-2 andL-3) in developing rice seeds have been identified [15] L-2(Type III) derives from 3-day-old seedlings [16] while RLL(Type I) is isolated from rice leaves [17] L-3 (Type III) is themajor component of these isozymes and accounts for 80ndash90 of their total activities [18] The lack of L-3 in DawDamleads to a decrease of lipid peroxidation and the alleviationof stale flavor accumulation by hexanal as well as pentanaland pentanol compounds during storage [19] UnfortunatelyDawDam could not be used for rice production due to itspoor agronomic characters [18] It is speculated that the lackof LOX-3 may affect the metabolism in the whole plant

In order to effectively improve rice grain storage qualitiesinactivation of some lipase andor LOXs should be achievedwithout compromising nutritional value and agronomicqualities RNAi could be an ideal method to achieve this goalbased on its applications on improving the nutritional qualityof various crops [20] Therefore the objectives of this studywere to select two candidate seedbran LOXs gene(s) developsuitable constructs for stable RNAi in rice and study thepotential benefits of silencing branseed-specific LOXs

2 Materials and Methods

21 Plant Materials The japonica rice cultivar ldquoLaGruerdquo wasused to clone target genes and develop transgenic plants Thetissues from DawDam Taipei-309 and transgenic lines wereused for gene expression analysis

22 Genomic DNA Extraction and PCR Analysis GenomicDNA was extracted from the leaf tissues of Taipei-309 seed-lings byNucleonPhytopure PlantDNAExtraction kit (Amer-sham Biosciences) according to the manufacturersrsquo protocol

Two Type III LOXs r9-LOX1 (accession numberAB099850) and L-2 (accession number X64396) and a leaf-specific LOX RCI-1 (accession number AJ270938) as well asRCI-1 (accession number AJ270938) were selected for studyInterestingly the L-3 (accession number E03480) sequencewas noted to be identical to the L-2 sequence (accessionnumber X64396) known to be distributed in rice embryo andhave C9 specific LOX activity [13] The nucleotide sequencesof L-2 and L-3 (Type III) were identicalThe primers for theseLOX genes were listed in Table 1 Gene sequences for invertedrepeats were amplified using thermostable proofreadingDNA polymerase from Stratagene (La Jolla CA USA) TheCACC sequence was added to the 51015840 end of forward primerfor facilitating directional incorporation into InvitrogenrsquospENTRD-TOPO entry vector PCR was performed by usingKOD-Plus-Neo (TOYOBO Japan) and then the productswere separated by 07 agarose gelThe target fragments werecollected using Qiangen gel purification kit

23 Construction of hpRNAi Vectors RNAi constructs wereprepared following the Gateway System protocol [21] Forconstructing hpRNAi vectors the target gene fragments werefirstly cloned into TOPO pENTR following the GatewaySystem protocol (Invitrogen Carlsbad CA)The transforma-tion of Agrobacterium tumefaciens EHA105 with the pANDAvector was performed using the Freeze and Thaw method[22] Agrobacterium-mediated transformation in rice wascarried out as described previously [23 24] withminormod-ifications [25]The positive control EHA105 was transformedwith pWHNG vector

24 Rice Transformation The transformation of Agrobac-terium tumefaciens EHA105 with the pANDA vector wasperformed using the Freeze andThawmethod [22] Agrobac-terium-mediated transformation in rice was carried out asdescribed previously [23 24] with minor modifications [25]The positive control EHA105 was transformed with pWHNGvector

Transformation was carried out using 21-day-old calligrown on B5 medium with 22mgL 2 4D After beingtreated with liquid cocultivation medium (YEP containingEHA105 at 600OD of 1) for 20min the calli were transferredto a solid cocultivation medium (B5 medium with 22mgL











3 Results













CDS L-2F CDS L-2R


2900bp




callus (d)


Transgenic plants






Mature seed

21 days


medium

3 days



15 days



15 days







B5-0

(a) (b)

(c)

(d)

(e)(f)(g)

(h)

(i)





100120583m ASB5 + 22mgL 2 4-D +






0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 A10 A11 A12

Relat

ive e

xpre

ssio

n le

vel



Plant tissue

(a)

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 B10 B11 B12

Relat

ive e

xpre

ssio

n le

vel



Plant tissue

(b)

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 C10

Relat

ive e

xpre

ssio

n le

vel

C11 C12



Plant tissue

(c)





4 Discussion



Products in pathway

Acetic acid

Hexanal

Nonanal

Naphthalene

GC RT (mins)

244

575

1511

1773

Control area counts

6866451

9019178

22363505

117817908


33524756

25689932

5738826

0


Abun

danc

e

400 600 800 1000 1200 1400 1600 1800ControlSample

Naphthalene

NonanalHexanal

Hexanol

Acetic acid

Time

14e + 0712e + 071e + 078e + 066e + 064e + 062e + 06

0

lowastlowast

lowast

lowast










5 Conclusion




Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











3 Results













CDS L-2F CDS L-2R


2900bp




callus (d)


Transgenic plants






Mature seed

21 days


medium

3 days



15 days



15 days







B5-0

(a) (b)

(c)

(d)

(e)(f)(g)

(h)

(i)





100120583m ASB5 + 22mgL 2 4-D +






0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 A10 A11 A12

Relat

ive e

xpre

ssio

n le

vel



Plant tissue

(a)

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 B10 B11 B12

Relat

ive e

xpre

ssio

n le

vel



Plant tissue

(b)

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 C10

Relat

ive e

xpre

ssio

n le

vel

C11 C12



Plant tissue

(c)





4 Discussion



Products in pathway

Acetic acid

Hexanal

Nonanal

Naphthalene

GC RT (mins)

244

575

1511

1773

Control area counts

6866451

9019178

22363505

117817908


33524756

25689932

5738826

0


Abun

danc

e

400 600 800 1000 1200 1400 1600 1800ControlSample

Naphthalene

NonanalHexanal

Hexanol

Acetic acid

Time

14e + 0712e + 071e + 078e + 066e + 064e + 062e + 06

0

lowastlowast

lowast

lowast










5 Conclusion




Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






CDS L-2F CDS L-2R


2900bp




callus (d)


Transgenic plants






Mature seed

21 days


medium

3 days



15 days



15 days







B5-0

(a) (b)

(c)

(d)

(e)(f)(g)

(h)

(i)





100120583m ASB5 + 22mgL 2 4-D +






0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 A10 A11 A12

Relat

ive e

xpre

ssio

n le

vel



Plant tissue

(a)

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 B10 B11 B12

Relat

ive e

xpre

ssio

n le

vel



Plant tissue

(b)

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 C10

Relat

ive e

xpre

ssio

n le

vel

C11 C12



Plant tissue

(c)





4 Discussion



Products in pathway

Acetic acid

Hexanal

Nonanal

Naphthalene

GC RT (mins)

244

575

1511

1773

Control area counts

6866451

9019178

22363505

117817908


33524756

25689932

5738826

0


Abun

danc

e

400 600 800 1000 1200 1400 1600 1800ControlSample

Naphthalene

NonanalHexanal

Hexanol

Acetic acid

Time

14e + 0712e + 071e + 078e + 066e + 064e + 062e + 06

0

lowastlowast

lowast

lowast










5 Conclusion




Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 A10 A11 A12

Relat

ive e

xpre

ssio

n le

vel



Plant tissue

(a)

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 B10 B11 B12

Relat

ive e

xpre

ssio

n le

vel



Plant tissue

(b)

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 C10

Relat

ive e

xpre

ssio

n le

vel

C11 C12



Plant tissue

(c)





4 Discussion



Products in pathway

Acetic acid

Hexanal

Nonanal

Naphthalene

GC RT (mins)

244

575

1511

1773

Control area counts

6866451

9019178

22363505

117817908


33524756

25689932

5738826

0


Abun

danc

e

400 600 800 1000 1200 1400 1600 1800ControlSample

Naphthalene

NonanalHexanal

Hexanol

Acetic acid

Time

14e + 0712e + 071e + 078e + 066e + 064e + 062e + 06

0

lowastlowast

lowast

lowast










5 Conclusion




Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Products in pathway

Acetic acid

Hexanal

Nonanal

Naphthalene

GC RT (mins)

244

575

1511

1773

Control area counts

6866451

9019178

22363505

117817908


33524756

25689932

5738826

0


Abun

danc

e

400 600 800 1000 1200 1400 1600 1800ControlSample

Naphthalene

NonanalHexanal

Hexanol

Acetic acid

Time

14e + 0712e + 071e + 078e + 066e + 064e + 062e + 06

0

lowastlowast

lowast

lowast










5 Conclusion




Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Competing Interests




Acknowledgments


References

















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Functional Characterization of 9-/13-LOXs ...downloads.hindawi.com/journals/bmri/2016/4275904.pdf · Here, RNA interference- (RNAi-) induced gene expression inhibition