Limonoids - Biosynthesis, Biochemistry and Analyis

7/30/2019 Limonoids - Biosynthesis, Biochemistry and Analyis

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BITTERNESS IN CITRUS

FRUIT-THE BIOCHEMISTRY,ANALYSIS AND

APPLICATIONS.

MANPREET KAUR

SAINI

21th march 2012


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CONTENTS OF PRESENTATION

BIOSYNTHESIS & BIOCHEMISTRY OF LIMONOIDS

EXTRACTION AND QUALITATIVE & QUANTITATIVE ANALYSIS OF CITRUS

BITTER PRINCIPLE.

GENE EXPRESSION AND TRANSCRIPTOME STUDIES

CLONING AND CREATION OF TRANSGENIC VARIETIES

APPLICATION OF THE BITTER PRINCIPLE


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PRIMARY AND SECONDARY METABOLITESMetabolites- compounds synthesized by organisms using enzyme-

mediated chemical reactions called metabolic pathways

PRIMARY METABOLITES

Essential to growth and development.

Present in all plants as are components or

products of fundamental metabolic

pathways or cycles.

SECONDARY METABOLITES

Not Essential to growth and development.

colored, fragrant, or flavorful compounds

typically mediate the interaction of plants

with other organisms.

EXAMPLES OF PRIMARY

METABOLITES

Energy rich fuel molecules, such as

sucrose and starch,Structural components such as cellulose,

informational molecules such a DNA

and RNA

Pigments such as chlorophyll.

EXAMPLES OF SECONDARY

METABOLITES

Alkaloids such as caffeine, nicotine, etc.

Terpenoids such as monoterpenes,diterpenes, triterpenes like Limonin and

tetraterpenes.

Phenolics such as flavonoids like naringin

and anthocyanins etc.

The main focus of this presentation will be triterpenoids mainly the Limonoids.


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INTRODUCTION TO BITTER PRINCIPLES

Kinnow mandarin, a hybrid of

Citrus nobilis and Citrus

deliciosa is considered one of

the major crops of Punjab.

But processing of Kinnow juice

faced formidable problems in

terms of bitterness and

delayed bitterness thusaffecting its consumer

acceptability.

Biochemical basis of bitterness

in kinnow:

bitterness due to flavonoids e.g.

naringin species related topumello. Threshold of bitterness

is 50 ppm.

delayed bitterness due to

limonoids e.g. limonin.

Threshold of bitterness is 6

ppm.

Critical reviews in

biotechnology(199

6)


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CITRUS BITTER PRINCIPLES IN DIFFERENT

PART OF THE FRUIT

Structure of citrus fruit showing concentration of limonoids and

flavonoids in different parts.

F- Flavedo, A- Albedo, SM-segment membrane, S- seeds, J- juiceKasetsart J. (Nat. Sci.) 43 : 28 - 36 (2009)


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BIOSYNTHESIS OF

LIMONOIDS

The Plant Cell, Vol. 7,

1015-1026, July (1995)

Acetyl Co A thiolase HMG Co A synthase

HMG Co A reductase

Mevalonate

kinase

PhosphoMevalonate

kinaseMVAP,Decarboxylase

GPP synthase

IPP Isomerase

FPP synthase

GGPP synthase

squalene

synthase

Terpene synthases

Prenyltransferase

s

MEVALONIC ACID PATHWAY AND TERPENOID SKELETON BIOSYNTHESIS( HMG Co A- 3 hydroxy 5 methyl glutaryl Co A, MVAP-Phosphomevalonate,MVAPP- 5-pyro Phosphomevalonate, IPP-Isopentyl Pyrophosphate, DMAPP-Dimethylallylpyrophosphate,

GPP-geranyl pyrophosphate, FPP- franesyl pyrophosphate, GGPP- geranyl geranyl pyrophosphate)


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BIOSYNTHESIS CONT.

SQUALENE

DEACETYLNOMILINIC ACIDNOMILIN

DEACETYLNOMILIN METHYL DEACETYLNOMILIC ACID

OBACUNONE

ICHANGENSIN(KETO) CALAMIN CYCLOCALAMIN

OBACUNOIC ACID

ICHANGIN ICHANGENSIN(KETAL)

LARL

LIMONIN DEOXYLIMONIN DEOXYLIMONIC ACID

LIMONOL

17 -D GLUCOPURANOSIDE

NOMILIN ACEYL

LYASE

HYDROLASE

DEHYDROGENASE

UDP-D-GLUCOSE:

LIMONOID GLUCOSYL

TRANSFERASE

Biol. Pharm. Bull. 29(2) 191201 (2006)


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BIOCHEMISTRY OF LIMONOIDS IN CITRUS

.

A group of highly oxygenated tetracyclic

triterpenoids in Rutaceae and Meliaceae

plant families causes delayed bitterness in

citrus and is a secondary metabolite.

Two forms in citrus:

1. Limonoid aglycones (LA) -- >50 isolated

from the Rutaceae (36 from Citrus & related

genera)

2. Limonoid glucosides (LG) -- 17 isolated

3. LARL and NARL- the precursors for

limonoid synthesis can also be considered

Relation between LA and LG1. LA: bitter, insoluble in water

2. LG: non-bitter, water-soluble

3. LA glucosidated to LG-during fruit maturation

and this is known as natural debittering.

Food review. Int, 12(4),(1996).


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LIMONOID AGLCONES J. Agric. Food Chem., Vol.55, No. 21, 2007 Review s


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LIMONOID GLUCOSIDES AND SYNTHETIC LIMONIN CARBOXYMETHOXIME

J. Agric. Food Chem., Vol.

55, No. 21, 2007 Review s


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SITES OF BIOSYNTHESIS OF LIMONOIDS

3 forms of limonoids

monolactone dilactone glucosides

Mature peel and fleshtissues In leavesand seeds

Limonin glucoside Nomilin glucoside


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ACCUMULATION OF LIMONOIDS

acetate, mevalonate, or farnesylpyrophosphate

Nomilin in stem phloem region

translocated to fruit

tissues, peels, seeds

and leaves

other limonoids


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MAJOR BIOSYNTHETIC GROUPS

Four groups of Limonoid Aglycones

1. Limonin group

2. Calamin group

3. Ichangensin group

4. 7-acetate limonoid group

Biosynthetic pathways of each group ofthese limonoids have been elucidated:


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THE LIMONIN BIOSYNTHETIC

GROUP in all citrus species, citrus hybrids and many non citrus members of family

rutaceae.

Limonin, nomilin, deacetylnomilin, Ichangin and obacunone are the major

limonoids


BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE LIMONIN GROUP


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CALAMIN BIOSYNTHETIC GROUP Found only in tissues of Fortunella and its hybrids like calamondin. And major limonoids

include calamin, retrocalamin, methyl iso-obacunoate diosphenol, 6-keto-7b-

deacetylnomilol and 6-keto-7b-nomilon.

6-keto-7b-nomilon contains structural features of both calamin and limonin groups-

represent a biosynthetic link between them.


BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE CALAMIN GROUP


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GROUP Found in tissues of Citrus ichangenesis and its hybrids Yuzu, Sudachi, Kabosu,Hanayu

and Ichang lemon, ichangenesin, deacetylnomilin, and deacetylnomilinic acid being the

major compounds.

present as ketal and keto group in chloroform solution but as ketal only in citrus.

Nomilin converted to deacetylnomilin by nomilin deacetylase, enzyme not found in any

other citrus species.


Nomilin

deacetlylase

BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE ICHANGENSIN GROUP


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THE 7-ACETATE LIMONOID BIOSYNTHETIC

GROUP Found only in tissues of Poncirus and its hybrids

This limonoid group includes 7a-obacunol, limonyl acetate [20] and 7a-obacunyl

acetate..

Food review. Int, 12(4),(1996).BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE 7-ACETATE lIMONOID GROUP


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DELAYED BITTERNESS

Most citrus fruits do not taste bitter if eaten fresh or if freshly squeezed juiceis consumed but within a few hours after juice extraction, the juice becomes

bitter. This phenomenon is generally referred to as delayed bitterness.

The two classes of chemical compound namely flavonoids and limonoids

were found responsible for bitterness in citrus juices. But there is a difference

between flavonoid and limonoid bitterness.

The fruits containing high flavonoids are bitter even when consumed as

fresh. The peel (rind) of the citrus fruit contain very high amount of flavonoids

like naringin, neohesperidine etc. making it highly bitter.

The limonoids are present in the form of non-bitter compound (limnoate - A-

ring lactone), which is converted to bitter limonin and other bitter limonoids in

the presence of enzyme limonoate-D-ring lactone hydrolase on storage.

Hence the fresh citrus juice does not taste bitter but turns highly bitter on

storage.


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FACTORS RESPONSIBLE FOR

DELAYED BITTERNESS

Cultivar

Location

Maturation time

Storage temperature

Acidic medium

Field Freeze injure

Mechanical damage

Initiates conversion of LARL to bitter

limonoid aglycone

Promotes enzme activity

With more time bitterness decreases.

Less the storage temperature, less the

bitterness as enzyme activity reduced

Different varieties has different level of

bitterness like in valencia orange and navel

orange.

Different temperature conditions affect the

bitternesss content of same fruit.


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MECHANISM OF DELAYED

BITTERNESS



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NATURAL LIMONOID DEBITTERING PROCESS

Limonin bitterness occurs in early to mid season harvested fruits only

Natural debittering process occurs when in late stages of fruit growth and

maturation limonoid aglycones were converted to their respectiveglucosides .

Limonoid glucosides contain one D- glucose molecule attached to the C-17

position of each corresponding aglycone via a beta- glucosidic linkage such

as limonin 17- beta D- glucopyranoside.

Enzyme involved is UDP-D_glucose transferase, isolated from citrus albedo

tissues, found only in mature fruit tissues and seeds .


ENZYMES INVOLVED IN BIOSYNTHESIS AND BIODEGRADATION


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ENZYMES INVOLVED IN BIOSYNTHESIS AND BIODEGRADATION

OF LIMONOIDS IN CITRUS

ENZYME GROUP NAME OF ENZYME SITE OF ACTION FUNCTIONS

E-1 Various enzymes ofterpenoid pathway likethiolase, synthase,

reductase, kinase,

isomerase, etc.

phloem region ofcitrus stem tissues biosynthesis of nomilinfrom acetate and

mevalonate

E-2 Enzymes like lyase,hydrolase,

dehydrogenase,

esterase, etc.

all citrus tissues

including leaves,

stems, fruit tissues,

fruit peels and seeds

biosynthesis of other

limonoids from Nomilin.

E-3 limonoid UDP- D-glucopyranoside

tranferase

seeds convert monolactones

to glucosides during

late stages of fruit

growth and maturation

E-4 limonoid D- ring lactone

hydrolase

seeds lactonization of the D-

ringconverts monolactones

to dilactones

E-5 limonoid 17- beta-D-glucopyranoside beta-

glucosidase

dormant seeds and

germinating seeds

limonoid biodegradation

catalyzes the hydrolysis

of limonoid glucosides

and liberates limonoids

and glucoseFood review. Int, 12(4),(1996)


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DEBITTRING METHODSDEBITTERING METHODS

column and batch

methods using

adsorbent and

ion exchange

resins

blend bitter

juice with non

bitter juice,

diluting out the

bitter taste

Creating new

citrus varieties

through

Genetic

engineering

Transgenic citrus varieties free from limoninbitterness

Three specific target enzymes

Insertion of one of the three gene

codings

Yields nonbitter fruits in

transgenic plants

Limonoid UDP-D-glucose

transferase

Limonoate Dehydrogenase

Nomilin deacetylase

Food review. Int, 12(4),(1996)

C t


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Cont

Nomilin deacetylaseLimonoid UDP-D-glucose

transferaseLimonoate Dehydrogenase

Converts limonoid aglycones

to glucosides

Convert limonin to nonbitter

limonoids

Convert nomilin to

deacetylnomilin

Enhancement of enzyme

activity through genetic

Engineering

reduces aglycone

concentration that reduces

delayed bitterness

In low level in citrus fruits

so isolated from bacteria

N terminal sequence

determined

Gene being from cDNA

library prepared from

bacterium

diverts biosynthetic

pathway of limonin

Nonbitter deacetylnomilin

accumlated instead of

limonin

Enzyme not isolated yet

Isolated from albedo tissues by a

combination of ammonium slfate

fractionation, affinity

chromatography,

and ion-exchange high-

performance liquid

chromatography (HPLC)

Isolated by ammonium

slfate fractionation, Blue

dye-ligand affinity

chromatography,

and ion-exchange HPLC

Food review. Int 12 4 1996


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EXTRACTION & QUALITATIVE &QUANTITATIVE ANALYSIS OF

CITRUS BITTER PRINCIPLE.


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Analytical Methods of Citrus

Limonoids

Thin-layer chromatography (TLC)

-- for limonoid detection

Nuclear Magnetic Resonance (NMR)

-- determination of limonoid structure

HPLC -- detection & quantification

Radioimmunoassaydetection &

quantification

HPLC-MS detection & quantification

EXTRACTION OF LARL & NARL FROM


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EXTRACTION OF LARL & NARL FROM

CITRUS JUICEJuice sample centrifuged at16000*g for 5min at 10 C.

Supernatant collected and filtered through 0.45 um filter.

Filtered liquid used to prepare three samples.

Juice(150 l)+CA internal standard

solution(75 l, 100 ppm)

Samples mixed and loaded(1 ml) onto strata X solid phase extraction columns

that was washed with MeOH(1 ml) and preconditioned with water(1 ml).

Thereafter column washed first with water(0.5 ml) and then CHCL3 (0.5ml) .

LARL finally eluted with solution B (CH3CN-MeOH-Water) (1 ml).

Juice (150 l)+internal

calibrator solution(15 l)

Juice (150 l)+

water (1.26 ml)

JOURNAL OF CHROMATOGRAPHY A, 1064(2005) 187-191


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EXTRACTION OF BITTER LIMONOIDS/ LIMONOID

AGLYCONES FROM CITRUS JUICE

Juice obtained by hand squeezing

Juice gently liquid/liquid extracted with CHCl3 (2*2.5 ml) for 30 s

3 l diluted extract was injected into LC-MS

2 ml juice transferred to test tube & 40 l internal standard added (167 ng/l)

CHCL3 extractscombined & 100 l of it removed.

It was evaporated to dryness and redissolved in MeOH or THF(300 l)

J. AGRIC. FOOD CHEMISTRY 2005, 51, 3709-3714


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EXTRACTION OF LIMONOID GLUCOSIDES

Wet samples (juice, molasses, wash

water) centrifuged at 16000 * g for 5 min

at 10 C.

Supernatant collected and filtered through

a 0.45 um filter.

Sample for injection prepared bycombining 75 l sample, 925 l water and

300 l MeOH and 200 l internal standard

solution

Samples analyzed by ESI-LC-MS

Solid samples (peel, seeds, etc) oven-dried

but pulp samples dried overnight at 60 C

and ground to pass 2mm mesh screen.

100 mg weighed into 10 * 50 mm cellulose

extraction thimbles.

Sample extracted overnight in soxhlet

extracter with 25 ml MeOH.

Diluted to 30 ml with MeOH

300 l of above extract + 700 l water + 200

l carminic acid solution(30 mg/l) added to

autosampler vial

Sample analyzed by ESI-LC-MS

J. AGRIC. FOOD CHEMISTRY 2005, VOL.49, NO 3, 2001


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GENE EXPRESSION AND

TRANSCRIPTOME STUDIES


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STUDY OF TRANSCRIPTOME CHANGES DURING

FRUIT DEVELOPMENT AND RIPENING FROM

DIFFERENT PARTS OF THE FRUIT.

STRUCTURE OF CITRUS

FRUIT

Braz. J. Plant Physiol., 19(4):333-362, 2007


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CITRUS FRUIT STRUCTURES TARGETED FOR ENZYMES

INDUCING NATURAL DEBITTERING IN CITRUS

SEED FLESH ALBEDO FLAVEDO

RNA ISOLATION AND CHARACTERIZATION AT DIFFERENT

STAGES OF FRUIT DEVELOPMENT

ANALYZING THE MECHANISM OF LIMONOID GLUCOSIDE

ACCUMULATION IN CITRUS

DEVELOPMENT OF TRANSGENIC PLANT WITH LOW LEVEL OF

BITTER LIMONOIDS TROUGH ENHANCEMENT OF NATURAL

DEBITTERING PROCESS

CITRUS FRUIT TRANSCRIPTOME SEQUENCING- HTS


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CITRUS FRUIT TRANSCRIPTOME SEQUENCING HTS

RNA ISOLATION AND cDNA LIBRARY PREPARATION

BIOINFORMATICS USAGE OF SOFTWARES FOR ILLUMINA GENOMEANALYSER DATA ASSEMBLY(Velvet, Oases, CLC GENOMICS)

TRANSCRIPTOME COMPARISON OF CITRUS FRUIT UNIGENE WITH

OTHER PLANT SPECIES

SEQUENCE ANALYSIS AND FUNCTIONAL ANNOTATION THROUGH GENE

ONTOLOGY(GO) CLASSIFICATION

TRANSCRIPT PER MILLION(TPM)-STATISTICAL CALCULATION BETWEENSAMPLES FOR DATA NORMALIZATION AND COMPARISON PURPOSE AND

ITS EXPRESSION PROFILING

DIFFERENTIAL EXPRESSION OF SELECTION OF GENES VALIDIATED BY

APPLYING REAL TIME QUANTITATIVE RT-PCR(q RT-PCR)Yu et al. BMC Genomics 2012, 13:10


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CITRUS FRUIT TRANSCRIPTOME SEQUENCING

USING MICROARRAY ANALYSIS

RNA ISOLATION AND cDNA LIBRARY PREPARATION

EST PROCESSING AND FUNCTIONAL ANNOTATIONMICROARRAY HYBRIDIZATION,SCANNING AND DATA ANALYSES

TRANSCRIPT PROFILING- first step in correlating gene expression with specific

biological processes, microarray being the high throughput method for it.

THE CONSTRUCTION OF A GENE- specific oligonucleotide chip based on

EST/genomic sequence data will expand microarray applications

METABOLOMICS AND PROTEOMICS DONE TO

1. correlate between compounds and/or proteins and a given trait or process.

2. to compare between cultivars displaying different traits, and to identify the compound(s)

and/or protein(s) associated with them.

3. to identify compounds of pharmaceutical, industrial and commercial values, such as

antioxidants, and unique aroma and flavor molecules

Plant Molecular Biology (2005) 57:375391


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CLONING AND CREATION OFTRANSGENIC VARIETIES


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DEVELOPMENT OF CITRUS VIRUS

VECTOR

sequencing plant genomes resulted in identification of large numbers of novel open reading

frames (ORFs) using large-scale functional genomic.

virus vectors for expression or silencing of plant genes.

Inoculation of plants with virus vectors a direct way to assay the function of specific genes

without the time consuming process of plant transformation and regeneration.

useful in woody plants like citrus, with long juvenile periods (up to 6-8 years).

complete sequence of a new virus, the citrus leaf blotch virus (CLBV) obtained.

used as a vector for expression or silencing genes in citrus plants because:

1.viruses like CTV being phloem limited, CLBV replicates in all citrus tissues.

2.accumulates mainly in meristematic tissues,offering an interesting model system to study

genes involved in growth and development of leaves and fruits;

3.monopartite genome of 8747nt containing three ORFs & easy to manipulate

4.mechanically transmissible to citrus & facilitate inoculation of engineered versions carrying the

foreign genes

CREATION OF TRANSGENIC CITRUS


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CREATION OF TRANSGENIC CITRUS

FREE FROM LIMONIN BITTERNESSGENETIC TRANSFORMATION OF CITRUS

EFFICIENT TRANSFORMATION IN CITRUS REQUIRED FOR TWO

MAIN REASONS:

TO IDENTIFY GENE FUNCTION

DEVELOPMENT OF NEW AND IMPROVED CITRUS VARIETIES

DEVELOPMENT OF TECHNOLOGIES FOR EFFICIENTTRANSFORMATION IN CITRUS

GENE REMOVAL

REMOVING GENES THAT ARE NO

LONGER USEFULUSEFUL WHEN THE PLANT IS

SMALL AND JUVENILE, NOT

REQUIRED IN THE MATURE TREE;

THIS WOULD PROVIDE THE

BENEFITS OF TRANSGENIC TRAITS

BUT RESULT IN NONTRANSGENIC

FRUIT.

GENE STACK ING.

MODIFYING MULTIPLE OR COMPLEX

CHARACTERISTICS OF A TREE

:INSERTION OF MULTIPLE GENES.

AND WOULD TAKE A LONG TIME.

GENE STACKING REQUIRES

TRANSFORMATION WITH MULTIPLE

GENES OR THE SEQUENTIAL

TRANSFORMATION..


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TECHNOLOGIES CONT

TECHNOLOGIES FOR GENETIC TRANSFORMATION OF CITRUS

NON-ANTIBIOTIC

SELECTION SYSTEMS

SELECT

TRANSFORMED

CELLS & TISSUES

WITHOUT USING

ANTIBIOTICS.

NEGATIVE (E.G.,

PHOSPHINOTHRICIN)AND POSITIVE (E.G.,

MANNOSE)

SELECTION

COMPOUNDS BE

DEVELOPED

LINKAGE GROUP

TRANSFORMATION.

TRANSFORMATION BY

LARGE DNA INSERTS

CONTAINING MULTIPLE

GENES IN LINKAGE

GROUPS

FACILITATING

POSITIONAL CLONING &FUNCTIONAL ANALYSIS

INTRODUCING MULTIPLE

GENES FOR SECONDARY

PRODUCT PATHWAYS

INTO CITRUS

.

GENE TARGETING.

TARGETING GENE

INSERTION BY

HOMOLOGOUS

RECOMBINATION INTO

THE CITRUS GENOME.

ALLOW DISRUPTION OF

GENE FUNCTION (KNOCK-

OUTS).

RESTORATION OF

FUNCTION OF DEFECTIVE

GENES (GENE THERAPY)

REPLACEMENT OF A

GENE WITH A NEW OR

ALTERED VERSION.


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TECHNOLOGIES CONT....

VIRAL-MEDIATED

TRANSFORMATION.

REQUIRE ANONPATHOGENIC VIRULENT

VIRAL VECTOR & A METHOD

TO DISPERSE THE VIRUS,

PRESUMABLY AN INSECT

VECTOR.

PHENOTYPE OF THE TREE

MODIFIED BY SIMPLY

DEPLOYING A DIFFERENT

ENGINEERED FORM OF THE

VIRUS.

TECHNOLOGIES FOR GENETIC TRANSFORMATION OF CITRUS

PROMOTER LIBRARY.

REPERTOIRE OF

PROMOTER & CIS-ACTINGELEMENTS CONTROLING

GENE EXPRESSION.

COORDINATION OF

APPROPRIATE EXPRESSION

LEVELS IN SPECIFIC

TISSUES OR CELL TYPES,

AT SPECIFIC

DEVELOPMENTAL STAGES,

AND UNDER VARIOUS

ENVIRONMENTAL

INDUCTION CONDITIONS.

MATURE TISSUE

TRANSFORMATION.

BYPASS THE LONGJUVENILITY PERIOD.

DIRECT TESTING

OF PUTATIVE GENE

CANDIDATES.


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APPLICATIONS OF

THE BITTER PRINCIPLES


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BIOLOGICAL ACTIVITY OF CITRUS

LIMONOIDS

Anticarcinogenesis Activity

(Inducers forGlutathione S-Transferase Activity)

Antifeedant Activity TaxonomicMarkers

Citrus limonoids posses furan

moiety attached to the D-ring at

the C-17 position that induces

GST activity.

Larvicidal effects.

AdUlt repellent and

oviposition deterrent

effect of limonoids.

Can be used as

taxonomic markers

as certainbiosynthetic

pathways are

unique to species

and genus.

Information as

such can be usedto evaluate existing

classification

schemes or to

modify those

schemes

Limonoids acts as

insecticides against

Colorado beetle, corn

earworm, fallarmyworm, spruce

budworm and

tobacco bud worm

Major limonoids with GST activity

includes nomilin,Obacuone,

Iso-obacunoic acid and ichangin

J. Agric. Food chem 2007, 55, 8285-8294


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Cont

The results of the rabbit study,expanded the anecdotal

evidence that secondary

metabolites, including

flavonoids and limonoids,

could function endogenously

to lower LDL cholesterol.

HYPOCHOLESTEROLEMIC

ACTIVITYANTIVIRAL ACTIVITY

limonin and nomilin have

also been evaluated for their

capacity to act as antiretroviral

agents.

They inhibit viral replication,

themechanism of action being

the inhibition ofin Vitro HIV-1

protease activity.

J. Agric. Food chem 2007, 55, 8285-8294

REFERENCES


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REFERENCES1. Douglas J. McGarvey and Rodney Croteau, Terpenoid Metabolism , The Plant Cell, Vol. 7, 1015-1026,

1995

2. Shin Hasegawa & Masaki Miyake, Biochemistry and biological functions of citrus limonoids, Food Rev.

Int,12(4),413-435,1996.

3. Gary D. Manners, Citrus limonoids: analysis, bioactivity and biomedical prospects, J.agric. Food Chem,

55, 8285-8294, 2007.

4. Munish puri, S.S. marwaha, R. M. Kothari, J.F. Kennedy ,Biochemical basis of bitterness in citrus fruit

juices and biotech approaches for debittering, Critical Reviews in Biotechnology, 16(2):145-155, 1996.

5. Andrew P. Breska, Audrius A. Zukas, Gary D. Manners, Detemination of LARL and NARL in citrus juices

by liquid chromatography-ESI MS, Journal of chromatography A, 1064, 187-191,2005.

6. Thomas K. Schoch, Garry D. Manners, Shin HasegawaAnalysis of Limonoid Glucosides from Citrus byESI LC MS, J.agric. Food Chem, 49 (3), 1102-1108, 2001.

7. Gary D. Manners, Andrew P. Breska , Thomas K. Schoch, Marlene B. Hidalgo, Analysis Of bitter

Limonoids in Citrus Juices by APCI and EI -LC-MS, J.agric. Food Chem, 51,3709-3714, 2003.

8. J. Forment etal, Development of a citrus genome-wide EST collection and cDNA microarray as

resources for genomic studies, Plant Molecular Biology (2005) 57:375391,2005.

9. Manuel Talon and Fred G. gmitter jr.,Citrus Genomics, International Journal of Plant Genomics, volume

2008.

10. Keqin Yu, Qiang Xu, Xinlei Da, Fei Guo, Yudon Ding, Xiuxin Deng, Transcriptome Changes during fruit

development and ripening of sweet orange(Citrus sinesis) BMC Genomics, 2012

11. M. Omura, M. Kita, . Endo-Inagaki, T. Moriguchi, R. Matsumoto, C. Suhayda, and Shin Hasegawa,

Genetic Evaluation and Modification of the Accumulation of Limonoids in Citrus, ACS Symposium

Series; American Chemical Society: Washington, DC, 2000.


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Limonoids - Biosynthesis, Biochemistry and Analyis