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CONTENTS OF PRESENTATION
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.
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 thus affecting its consumer acceptability. Biochemical basis of bitterness in kinnow:
bitterness due to flavonoids e.g. naringin species related to pumello. 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)
Structure of citrus fruit showing concentration of limonoids and flavonoids in different parts.
F- Flavedo, A- Albedo, SM-segment membrane, S- seeds, J- juice
Kasetsart J. (Nat. Sci.) 43 : 28 - 36 (2009)
BIOSYNTHESIS OF LIMONOIDS
Acetyl Co A thiolase HMG Co A synthase HMG Co A reductase PhosphoMevalonate kinase MVAP,Decarboxylase Mevalonate kinase
FPP synthase
squalene synthase GGPP synthase
Terpene synthases
BIOSYNTHESIS CONT.
SQUALENE
NOMILIN ACEYL LYASE
DEACETYLNOMILINIC ACID
DEACETYLNOMILIN ICHANGENSIN(KETO) METHYL DEACETYLNOMILIC ACID CALAMIN CYCLOCALAMIN
NOMILIN
HYDROLASE
LARL
DEHYDROGENASE
LIMONIN LIMONOL
DEOXYLIMONIN
UDP-D-GLUCOSE: LIMONOID GLUCOSYL TRANSFERASE
DEOXYLIMONIC ACID
17 -D GLUCOPURANOSIDE
Biol. Pharm. Bull. 29(2) 191201 (2006)
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
LIMONOID AGLCONES
Limonin glucoside
Nomilin glucoside
ACCUMULATION OF LIMONOIDS
acetate, mevalonate, or farnesyl pyrophosphate
Nomilin
other limonoids
1. Limonin group 2. Calamin group 3. Ichangensin group 4. 7-acetate limonoid group Biosynthetic pathways of each group of these limonoids have been elucidated:
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
Found only in tissues of Fortunella and its hybrids like calamondin. And major limonoids include calamin, retrocalamin, methyl iso-obacunoate diosphenol, 6-keto-7bdeacetylnomilol and 6-keto-7b-nomilon. 6-keto-7b-nomilon contains structural features of both calamin and limonin groupsrepresent a biosynthetic link between them.
THE ICHANGENSIN BIOSYNTHETIC 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
DELAYED BITTERNESS
Most citrus fruits do not taste bitter if eaten fresh or if freshly squeezed juice is 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 - Aring 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.
Cultivar Location Maturation time Storage temperature Acidic medium Field Freeze injure Mechanical damage
Different varieties has different level of bitterness like in valencia orange and navel orange. Different temperature conditions affect the bitternesss content of same fruit.
Less the storage temperature, less the bitterness as enzyme activity reduced
Promotes enzme activity
E-1
Various enzymes of terpenoid pathway like thiolase, synthase, reductase, kinase, isomerase, etc. Enzymes like lyase, hydrolase, dehydrogenase, esterase, etc. limonoid UDPglucopyranoside tranferase D-
E-2
all citrus tissues including leaves, stems, fruit tissues, fruit peels and seeds seeds
E-3
convert monolactones to glucosides during late stages of fruit growth and maturation lactonization of the Dring converts monolactones to dilactones limonoid biodegradation catalyzes the hydrolysis of limonoid glucosides and liberates limonoids and glucose
Food review. Int, 12(4),(1996)
E-4
seeds
E-5
DEBITTRING METHODS
DEBITTERING METHODS
column and batch methods using adsorbent and ion exchange resins
blend bitter juice with non bitter juice, diluting out the bitter taste
Transgenic citrus varieties free from limonin bitterness Limonoid UDP-D-glucose transferase Three specific target enzymes Limonoate Dehydrogenase Nomilin deacetylase Insertion of one of the three gene codings
Cont
Limonoid UDP-D-glucose transferase Limonoate Dehydrogenase Nomilin deacetylase
to
Isolated from albedo tissues by a combination of ammonium slfate fractionation, affinity chromatography, and ion-exchange highperformance liquid chromatography (HPLC)
Isolated by ammonium slfate fractionation, Blue dye-ligand affinity chromatography, and ion-exchange HPLC
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
Food review. Int, 12(4),(1996)
Analytical Methods of Citrus Limonoids Thin-layer chromatography (TLC) -- for limonoid detection Nuclear Magnetic Resonance (NMR) -- determination of limonoid structure HPLC -- detection & quantification Radioimmunoassay detection & quantification HPLC-MS detection & quantification
CITRUS JUICE
Juice sample centrifuged at16000*g for 5min at 10 C.
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) .
2 ml juice transferred to test tube & 40 l internal standard added (167 ng/l)
Sample for injection prepared by combining 75 l sample, 925 l water and 300 l MeOH and 200 l internal standard solution
300 l of above extract + 700 l water + 200 l carminic acid solution(30 mg/l) added to autosampler vial
STUDY OF TRANSCRIPTOME CHANGES DURING FRUIT DEVELOPMENT AND RIPENING FROM DIFFERENT PARTS OF THE FRUIT.
CITRUS FRUIT STRUCTURES TARGETED FOR ENZYMES INDUCING NATURAL DEBITTERING IN CITRUS
SEED
FLESH
ALBEDO
FLAVEDO
DEVELOPMENT OF TRANSGENIC PLANT WITH LOW LEVEL OF BITTER LIMONOIDS TROUGH ENHANCEMENT OF NATURAL DEBITTERING PROCESS
BIOINFORMATICS USAGE OF SOFTWARES FOR ILLUMINA GENOME ANALYSER DATA ASSEMBLY(Velvet, Oases, CLC GENOMICS)
TRANSCRIPT PER MILLION(TPM)-STATISTICAL CALCULATION BETWEEN SAMPLES 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
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
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;
GENE STACKING. 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..
GENE REMOVAL REMOVING GENES THAT ARE NO LONGER USEFUL USEFUL 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.
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 (KNOCKOUTS). RESTORATION OF FUNCTION OF DEFECTIVE GENES (GENE THERAPY) REPLACEMENT OF A GENE WITH A NEW OR ALTERED VERSION.
TECHNOLOGIES CONT....
TECHNOLOGIES FOR GENETIC TRANSFORMATION OF CITRUS
PROMOTER LIBRARY. REPERTOIRE OF PROMOTER & CIS-ACTING ELEMENTS 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 LONG JUVENILITY PERIOD. DIRECT TESTING OF PUTATIVE GENE CANDIDATES.
VIRAL-MEDIATED TRANSFORMATION. REQUIRE A NONPATHOGENIC 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.
Citrus limonoids posses furan moiety attached to the D-ring at the C-17 position that induces GST activity.
Major limonoids with GST activity includes nomilin,Obacuone, Iso-obacunoic acid and ichangin
Limonoids acts as insecticides against Colorado beetle, corn earworm, fall armyworm, spruce budworm and tobacco bud worm
Can be used as taxonomic markers as certain biosynthetic pathways are unique to species and genus. Information as such can be used to evaluate existing classification schemes or to modify those schemes
Cont
HYPOCHOLESTEROLEMIC ACTIVITY ANTIVIRAL ACTIVITY
The results of the rabbit study, expanded the anecdotal evidence that secondary metabolites, including flavonoids and limonoids, could function endogenously to lower LDL cholesterol.
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 of in Vitro HIV-1 protease activity.
REFERENCES
1. 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 by ESI 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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