Lecture 3 a Genomics Approach to Flavour

8/3/2019 Lecture 3 a Genomics Approach to Flavour

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The genomics revolution:

Opening up a fruit

Robert Schaffer

BIO340


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Genomics

! Large scale analysis of genes and theirexpression

! Microarrays! mRNA seq (deep sequencing)! qPCR! There are often multiple copies of a gene

within a genome


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Microarrays

! Hybridisation technology! Fix known genes to a solid surface! Test abundance of genes by labelled mRNA

population! Mature technology! Need to know the sequence of the genes

you are assaying


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mRNA seq (deep sequencing)

! Sequence based gene expression assay! Isolate RNA convert to cDNA! Sequence

! Frequency count of numbers of transcripts! Large numbers highly expressed! Low numbers low expression

! Pricy


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What is flavour?

! Sum of a complex interaction between taste receptors,the ortho- and retronasal olfactory systems, mouthtexture, and visual appearance

! Taste receptors respond to sugars (principally glucoseand fructose), acids (citric, malic, and ascorbic) andglutamate

! Volatile chemicals impart the distinctive fruit flavour foreach fruit


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Flavour in Tomato

! Over 400 volatiles have been detected in tomato! Not all volatiles are equal, flavour depends on volatile

activity (threshold for detection)

!

1520 are made in sufficient quantities to have animpact on human perception


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A selection of tomato volatiles

Conc Odor threshold Log Odor activity

cis-3-Hexenal 12,000 0.25 3.7

b-ionone 4 0.007 2.8

Hexanal 3,100 4.5 2.8

b-Damascenone 1 0.002 2.7

1-Penten-3-one 520 1 2.7

2-Phenylethanolx 1,900 1,000 0.3

3-Methylbutanol 380 250 0.2

Pentanol 120 4,000 1.5

Pseudoionone 10 800 1.9

Isobutyl cyanide 13 1,000 1.9

Hexanol 7 500 1.9

Epoxy-b-ionone 1 100 2.0


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Attractant properties of aroma

Tomato fruits produce a volatile emission profile that is both attractive to humans

and an indicator of ripeness. Of the more than 400 volatiles emitted by tomato fruits,only a small number, almost all of which are derived from essential human nutrients,are detected and integrated into a preferred volatile aroma. This pattern of volatileemissions is mutually beneficial. Thus, volatile emissions are both positive indicatorsfor the presence in the fruit of compounds with positive health benefits andattractants that promote seed dispersal.

Goff and Klee Science 2006


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Genomics approach to findingtomato volatiles

! Fruit development series of Tomato! Identify genes that change and those

regulated by ethylene! Alba et al 2005

! These studies generate large quantities ofdata that can be mined for aroma relatedgenes


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Fruit volatile traces

Apple

Kiwifruit

Capegooseberry

Necterine

Passionfruit


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Role of models?

! Models cannot answer some traits! Need to be addressed in the species of interest! Genomics approaches

! Recent advances in sequencing technologies havemeant that molecular biology questions can beanswered in non model systems

! An EST approach allows the identification of aromabased genes based on homology

! Expression analysis of these genes can narrowcandidate genes that can be subsequently tested


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KEGG:- KyotoEncyclopedia of Genesand Genomes


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Case study Apple study at Plantand Food Research

! A genomics approach to volatile analysis inApples

! EST sequencing project late 90s 150K ESTsfrom many stages in apple development

! These represent ~20,000 unique genes! Microarray development 16,000 oligonucleotides

synthesised to unique regions on the gene(focussed on 3 end)

! Gene expression analysis of ethylene inducedripening of the ACO oxidase knockout


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ESTERS

Fatty Acidbiosynthesis

Amino Acidbiosynthesis

Enzymic steps 9 4

Genes* 31 13

Microarray oligos 19 11

Data Mining Volatile Genes

Enzymic steps

Genes*

9

71

Microarray oligos 65

TERPENES

9

17

17

!-farnesene

PHENYLPROPANOIDS

8

46

45

estragole

Total candidate volatiles206


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Time (h)

+ C2H4

100ppm C2H4

!!!! !!!!! !

Skin

Cortex !!!! !!!!! !

Rep1

Rep2

Rep1

Rep2

0 4 18 96 192

- C2H4

! !! !

! !! !

0 4 18 96 192

Time (h)

Skin

Cortex

Rep1

Rep2

Rep1

Rep2

Gene expression analysis


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Volatiles induced by ethylene

18

96

192

2-methylbutyl3-oxobutanoate ethyl hexanoate

ethyl acetatemethyl butanoatehexenoic acid

pentyl butanoatetoluene

hexyl 2-methyl butanoatebutyl heptanoate

1-butanolpropyl acetateheptyl acetate

estragolebutyl propanoate

butyl acetate2-ethyl hexyl acetate2-methylbutyl acetate

2-methylpropyl acetatehexyl propanoate

hexyl acetatehexanol

pentyl acetate or 3mba(Z,E) alpha-farnesene

butyl formatepropyl hexanoate

(E,E) alpha-farnesenebutyl 2-methyl butanoate

pentyl hexanoatebutyl hexanoatebutyl butanoate

hexyl hexanoate

0 20 40 600.0 1.0

Maximum levels (ng/g)0 4 C

esters!-farneseneestragole


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Selection of genes that change

! ANOVA models! Tissue types (Skin:Cortex)! Time course (0,4,18,96,192 hrs)! Tissues * time

! Used a FDR threshold of 0.05 to select genes! 1923 genes selected! Group genes by expression pattern


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04

18

96

192

C

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

Gene expression in skin

Rapid induction

Expression

0 4 18 96 192 C

Time

0 4 18 96 192 C

Time

Inhibition

0 4 18 96 192 C

Time

Induction


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EstersFatty acid biosynthesis

Fatty acid degradation

Isoleucine biosynthesis

Isoleucine degradation

Lipoxygenase (EC 1.13.11.12)

13 hydroperoxidelinoleic acid

13 hydroperoxidelinolenic acid

Hydroperoxide lyase (EC 4.2.1.92)

Hexen-3-alHexanal

Branched chain aminotransferase(EC 2.6.1.42)

2-oxo-3-methylpentanoic acid

Decarboxylase(EC 4.1.1.x)

2-oxoacid dehydrogenasecomplex (EC 1.2.4.4, EC1.8.1.4, EC 2.3.1.168)

2-methylbutanal

Il12Es1

Es1a-d

Es2

0.0 0.2 0.4 0.6 0.8 1.0

esters

acidsalcohols

Alcohol dehydrogenase(EC 1.1.1.1)

Hex-3-enolHexanol

Alcohol acyl transferase(EC 2.3.1.x)

Hex-3-enyl acetateHexyl acetate

2-methylbutanoyl CoA2-methylbutanol

2-methylbutyl acetate Hexyl 2-methyl butanoate

Aldehyde dehydrogenase(EC 1.2.1.3)Es3

Es4

Es50 418

96

192

C

Time


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phenylpropanoid

Estragol biosynthesis

estragole

O-methyl transferase (EC 2.1.1.x)S-adenosyl-L-methionine carboxylMethyltransferase (EC 2.1.1.x)

Phenylalanine ammonia lyase(EC 4.3.1.5)

Cinnimate-4-hydroxylase(EC 1.4.13.11)

4-Coumarate-CoAligase (EC 6.2.1.12)

Cinnamoyl-CoA reductase (EC1.2.1.44)

Cinnamyl-alcohol dehydrogenase(EC 1.1.1.95)

Unkown Dehydratase ?

Phenylalanine

Cinnamate

4-coumaric acid

Coumarate-CoA

Coumaroyl aldehyde

Coumaroyl alcohol

Chavicol

Estragole

P1

P2

P4

P5

P6

P7

caffeic acidP3

sesquiterpene

!-farnesene biosynthesis

!-farnesene

!-farnesene

Acetyl-CoA acetyltransferase(EC 2.3.1.9)

HMG-CoA synthase(EC 2.3.3.10)

HMG-CoA reductase(EC 1.1.1.34)

Mevalonate kinase(EC 2.7.1.36)

Phosphomevalonate kinase(EC 2.7.4.2 )

Mevalonate 5-diphosphate decarboxylase(EC 4.1.1.33)

Farnesyldiphosphate synthase(EC 2.5.1.10)

!-farnesene synthase

Acetyl CoA

Acetoacetyl CoA

3-hydroxy-3-methylglutaryl CoA

Mevalonate

Mevalonate-5-phoshate

Mevalonate-5-diphoshate

FFP

Isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2)

IPP DMAP

T1

T2

T3

T4

T5

T6

T7

T8

T9


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Esters biosynthesis

! 24 different ester compounds weredetected in the volatile traces

! Only one acyl transferase (AT1) detectedas increasing in expression?

Basic chemistry

Alcohol + Acid-CoA Ester + Water

Acyl transferase

(eg. Acetyl-CoA)


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Testing the activity of the AT1

! Clone the Apple gene by PCR! Over express in E.coli! Crude protein isolation! Test with different combinations of alcohol

plus acid (CoA conjugated) precursors


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Results from the enzyme test

Souleyre et al. 2005 FEBs journal


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Function of AT1

! Promiscuous enzyme! Enzyme can synthesise many esters

suggesting that the limitation of aroma

compounds maybe in the precursormolecules


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Volatiles induced by ethylene

18

96

192

2-methylbutyl3-oxobutanoate ethyl hexanoate

ethyl acetatemethyl butanoatehexenoic acid

pentyl butanoatetoluene

hexyl 2-methyl butanoatebutyl heptanoate

1-butanolpropyl acetateheptyl acetate

estragolebutyl propanoate

butyl acetate2-ethyl hexyl acetate2-methylbutyl acetate

2-methylpropyl acetate

hexyl propanoatehexyl acetate

hexanolpentyl acetate or 3mba(Z,E) alpha-farnesene

butyl formatepropyl hexanoate

(E,E) alpha-farnesenebutyl 2-methyl butanoate

pentyl hexanoatebutyl hexanoatebutyl butanoatehexyl hexanoate

0 20 40 600.0 1.0

Maximum levels (ng/g)0 4 C

esters!-farneseneestragole


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Minimum ethylene needed for ripening

ACOas Continuous ethylene

treatment

0.01 ppm

1 ppm

10 ppm

0.1 ppm

100 ppm

1000 ppm

0.0 ppm

Full climacteric apples(500ppm)

Maximum level of detectedethylene in ACOas lines(0.019ppm)


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Dose response curves of differentvolatiles


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Regulation of flavour in apples


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Flavour in apples

! In the quest to get better textured apples that store better! Old varieties good flavour

! Coxs high ethylene poor storage! Reduced ethylene varieties

!Rely totally on the Acid/Sugar balance for flavour! Granny Smith! Breaburn

! Exception is the Jazz apple, good texture and goodflavour

!

Creating new flavours in apple! Breeding lines using diverse parents for cross more likely to yieldnew flavours


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Diversity of volatiles in a RoyalGala x Granny Smith cross

Rowan et al 2009


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Would you follow the same path forcape gooseberry?

! What has changed?! More routine measures for selecting genes that

change

! Would you bother with microarrays at all?! Sequencing data is considerably cheaper

Deep sequencing approach?! Now can generate ~10M sequence reads for ~$9K

! Web based tools are improving all the time! New ways of validating enzymic function! (Hellens et al. 2005 Plant methods)


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MapmanAligning expression patterns on known biosynthetic pathways


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Themes

! Acid sugar balance! Aroma volatiles! Difference between different fruit! Pools of precursors often synthesised by single enzyme! Going beyond models, have to interrogate the plant ofinterest! Genomics have allow us to go beyond models! In apples

! Genes controlling the synthesis are regulated at the lastenzymatic step suggesting pools are present ready to go inmature fruit

Lecture 3 a Genomics Approach to Flavour

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