This document summarizes a presentation on the plant enzyme chalcone synthase (CHS). It discusses that CHS is a key enzyme in the phenylpropanoid pathway that produces flavonoids. It describes the localization and regulation of CHS gene expression and activity, which can be controlled by metabolic feedback, turnover rates, and transgenic methods. CHS expression and activity increases in response to stresses like UV light and pathogen infection, inducing the production of protective phytoalexins.

1. 9/6/2014 Department of Plant Biotechnology 1

2. 9/6/2014 Department of Plant Biotechnology 2
BY:
AVINASH SHARMA
PALB-3235
Sr M.Sc.

3. Contents:-
• Introduction
• Control of CHS Activity
• CHS localization
• Regulation of CHS Gene Expression
• CHS activity in Plant Resistance
• Case Study
• Seminar Conclusions
• References
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4. Introduction:-
• Chalcone synthase (CHS) belongs to a family of polyketide synthase enzymes
(PKS) known as type III PKS.
• Type III PKSs are associated with the production of chalcones.
• First bactreia Streptomyces griseus were observed PKS III Chalcone synthase and
in Plants Chalcone synthase were first observed in the barley leaves.
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5. Contd:-
• CHS gene expression is induced under stress conditions such as UV light and
bacterial or fungal infection.
• Chalcone synthase produce chalcone during Phenylpropanoid pathway and
Flavonoid pathway therefore chalcone synthase are the key enzyme.
• Chalcone synthase enzyme produce flavonoids like lignin, suberin and
isoflavonoids like genistein, wighteone and luteon and protect from the attack
of pathogen and UV light.
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6. Phenylpropanoid Metabolic Pathway:-
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7. Contd:-
Enzyme names are abbreviated as follows:-
• PAL- phenylalanine ammonia-lyase; C4H- cinnamate 4-hydroxylase;
• 4CL- 4-coumarate: CoA ligase ; CHS- chalcone synthase
• CHI- chalcone isomerase; IFS- isoflavone synthase
• F3'H- flavonoid 3'-hydroxylase ; F3'5'H flavonoid 3',5'-hydroxylase
• F3H- flavanone 3-hydroxylase; DFR- dihydroflavonol-4reductase
• ANS- anthocyanidin synthase also called LDOX, leucoanthocyanidin dioxygenase);
• UFGT- UDP-flavonoid glucosyltransferase; BA2H- benzoic acid 2-hydroxylase
• C3H- p-coumarate 3 hydroxylase ; COMT- caffeic O-methyltransferase
• F5H- ferulic acid 5-hydroxylase ; CCR, cynnamoyl CoA reductase
• CAD, cynnamyl alcohol dehydrogenase
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8. CHS localization and determination:-
• CHS proteins found in the different plant organs. For example:-
• CHS protein in Buckwheat (Fagopyrum esculentum) hypocotyls is located in the
cytosol and associates with the cytoplasmic face of the rough endoplasmic
reticulum (rER), but not with nuclei, plastids, mitochondria, Golgi, or
tonoplasts.
• CHS(chalcone synthetase) and CHI(chalcone isomerase) are found in the
Arabidopsis roots and also found in epidermal and cortex cells of the
elongation zone and the root tip.
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9. Contd:-
Flavonoids found in the nucleus of different species such as:-
Arabidopsis thaliana (thale cress, mouse-ear cress or arabidopsis);
Brassica napus (Rape, Oilseedrape, Rapa, Rappi, Rapeseed);
Flaveria chloraefolia (Yellowtops);
Picea abies (Norway spruce);
Tsuga Canadensis (Eastern hemlock or Canadian hemlock); and
Taxus baccata (Conifer).
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10. Contd:-
Determination of CHS:-
• Immuno gold-labeling or immuno gold stains in the grape berry plant and the
site of the CHS. CHS was found in the:-
 Rough Endoplasmic Reticulum (RER);
 Cytoplasm of the skin cells;
 Cell wall;
Cells of developing Grape berry;
 Plastid;
 Vacuole and
 Vacuole membrane (tonoplast).
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11. Control of CHS activity:-
• Phenylpropanoid pathway regulated by the activity of CHS.
• CHS activity was first described in 1972 in extracts of parsley (Petroselinum
crispum).
Chalcone synthase activity control following ways:-
A) Metabolic control
B) Control of CHS turnover
C) Control of CHS through trans-genes
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12. A) Metabolic control:-
• Flavonoid pathway products like naringenin, chalcone naringenin and the other
end products of CoA esters inhibit the activity chalcone synthase non
competitively.
• Metabolic products like naringenin and chalcone narigenin can inhibit CHS at
100 μM.
• Different metabolic product like flavonoids and chalcones inhibit the activity of
CHS in severals crops. For example:-
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13. Contd:-
• For example:-
 Flavonoids luteolin and apigenin are inhibitory to rye CHS.
In carrot naringenin and chalcone narigenin can inhibit CHS at 100 μM.
In cytosol flavonoid concentration are more then flavonoid blocks the activity
of CHS.
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14. B) Control of CHS turnover:-
• UV light and biotic elicitors induce the Flavonoid biosynthetic pathway at the
transcriptional level and that CHS is not turn to produce product.
• Studies on parsley cell cultures showed that UV light was given to the
chalcone synthetase (CHS) and resulted that the activity of enzymes decayed
with a half-life of 6 h, whereas inactive enzyme decayed more slowly with a
half-life of 18 h.
• The accumulation of flavonoid end products was got limiting step(s) in
flavonoid biosynthesis then CHS activity may not reflect in vivo.
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15. C)Control of CHS through trans-genes:-
• Activity of CHS can be controlled by antisense or sense genes.
• Transgenic Petunia, the antisense construct was able to inhibit expression of
CHS genes , inhibition of anthocyanin production to give completely acyanic or
patterned flowers.
• Homologous pairing between the transcripts of sense CHS genes and
transcripts of antisense CHS gene to form double stranded RNA that is very
rapidly degraded, thus inhibiting the activity of CHS transcript RNA.
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16. Regulation of CHS gene expression:-
• Regulation of CHS gene are induced by light/UV light and response of
phytopathogens.
H-Box (CCTACC), G-Box
(CACGTG), a/a2 regulation
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17. Contd:-
• CHS promoter contains regulatory nucleotide sequence CACGTG known as G-
box, which has been found to be important in the response to light/UV light.
• Another CHS promoter regulatory nucleotide sequence (CACGTG) known as G-
Box and this box are involved in Transcription.
• In Phaseolus vulgaris CHS15 gene promoter contains Box I, Box II, Box III, Box IV
or three copies of H-box (CCTACC). G-box and H-box are together required for
light inducibility.
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18. Contd:-
• Silencer element located between positions -326 and -173 of the CHS15
promoter.
• CHS15 gene in response to fungal elicitors and glutathione are located in the
130 bp region of the promoter.
• CHS gene expression is reflected when transcription take place and the RNA
polymerase II must attach to specific DNA sequences in the CHS promoter in the
vicinity of the TATA box and must be activated by transcription factors binding
and response to upstream in the promoter.
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19. Contd:-
• Barley leaves inoculated with the fungus Blumeria graminis f.sp. hordei (Bgh) (downy
mildew). HvCHS2 transcripts accumulate strongly in barley leaves in comparison to
naringenin-CHS (CHS1).
• Soybean synthesis of lignin/suberin through Phenylpropanoid pathway after Psg-avrB
gene inoculation which response early to Pseudomonas syringae pv. Glycinea (bacterial
blight).
• A cDNA encoding a chalcone synthase was isolated from the leaves of Polygonum
minus (knotweed, knotgrass) by rapid amplification of cDNA ends (RACE) and
designated pmCHS.
• qRT-PCR showed that pmCHS was most highly expressed in the roots, showing a 10-
fold increase compared to leaves and a 15-fold increase compared to stems.
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20. CHS activity in Plant resistance:-
• CHS is quite commonly induced in different plant species under different forms
of stress like
 UV light,
 Wounding,
 Microbial Pathogens
resulting in the production of compounds that have e.g. antimicrobial activity
(Phytoalexins), insecticidal activity, and antioxidant activity or quench UV light
directly or indirectly.
• Current knowledge about regulation of CHS in plant pathogen resistance is
presented in Table
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21. Chalcone synthase expression in plant under stress conditions:-
Sl.
No.
Host Crop Pathogen/stresses Metabolites
1. Petroselinum crispum Parsley UV Flavonoids
2. Hordeum vulgare Barley Blumeria graminis
Erysiphe graminis
UV
Flavonoids
3. Glycine max Soybean Pseudomonas
syringae pv glycinea,
Phytophthora
megasperma
f. sp. Glycinea.
Flavonoids
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22. Contd:-
Sl. No. Host Crop Pathogen/stresses Metabolites
4. Pinus sylvestris Scots pine UV-B Phenolic
compounds,
flavonoids, catechin
5. Medicago truncatula (Barrel
Clover) Medicago sativa
Alfalfa, Barrel Clover Glomus versiforme Isoflavonoid
6. Daucus carota Carrot UV, Pythium
aphanidermatum
Anthocyanin
7. Brassica rapa Turnip UV Anthocyanin
8. Sorghum bicolor Sorghum mesocotyl,
juvenile sorghum
tissues
Colletotrichum
graminicola ,
Helminthosporium
maydis
3-
Deoxyanthocyanidins
, apigeninidin
luteolinidin
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23. Contd:-
Sl. No. Host Crop Pathogen/stresses Metabolites
9. Picea abies Norway spruce Ceratocystis polonica,
Ophiostoma
polonicum and
wounding
Catechin
10. Arabidopsis thaliana Thale cress
Low temperature,
UV-B, UV-A, and blue
Light
Anthocyanins
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24. Phytoalexins:-
• Phytoalexins are produced by plants in response to microbial attack ( biotic and
abiotic elicitors).
• Accumulation of flavonoids and isoflavonoids in response to pathogen attack is
seen in many plant species, and their importance as antimicrobial phytoalexins
is well established . For example:-
a) Isoflavonoids were increased in Lupinus luteus (annual plant) after infection
with Fusarium oxysporum (Fusarium wilt) such as genistein, wighteone and
luteon.
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25. Contd:-
b) The isoflavones, daidzein, genistein and glycitein, in soybean were strongly
increased after infection by Sclerotinia sclerotiorum (stem rot).
• Stilbenes are known as the phytoalexins in peanut and grapes. There is also
evidence that stilbene synthase (STS) has developed from CHS several times in
the evolution.
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26. Light protection:-
• Phenolic compounds like flavonoids strongly absorb UV light and thus are able
to protect plants from DNA damage caused by UV.
• Anthocyanins belong to a class of flavonoids that accumulate in leaves and
stems as plant in response to light intensity .
• Expression of CHS genes is known to be regulated by light through a
photoreceptor-mediated mechanism.
• In (binneal plant) parsley cell culture suggested that a UV-B light receptor, a
blue light receptor and phytochrome may all play a role in light- induced CHS
expression.
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27. CASE STUDY
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* Correspondence: hatem.boubakri@cbbc.rnrt.tn 1Laboratoire de Physiologie Moléculaire des Plantes,
Centre de Biotechnologie de Borj-Cédria, 2050, Hammam Lif, Tunisie 2Unité Mixte de Recherche 1131,
Université de Strasbourg /INRA-Colmar, 28 Rue de Herrlisheim, F68021, Colmar, France

29. Introduction:-
• Thiamine (vitamin B1) induce resistance against Plasmopara viticola pathogen
(downy mildew of leaf) in grape vine “Chardonnay cv” plant.
• Thiamine (vitamin B1) induce the Phenylpropanoid pathway genes which
derived phytoalexins and induce resistance against Plasmopara viticola
pathogen.
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30. Material and methods:-
Plant material and growth conditions:-
• Vitis vinifera “Chardonnay cv” plants were obtained from herbaceous cuttings
and cultivated in the pots and
• Vitis vinifera “Chardonnay cv” plants (grapevine plant) were kept in the
glasshouse and provide growth conditions like temperature 24°C, 16 h light and
8 h dark photoperiod and 70% RH.
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31. contd:-
Pathogen:-
• Vitis vinifera “Chardonnay cv” plants leaves were collected and placed in the
petriplates and petriplates contains infectious propagule sporangia of
Plasmopara viticola (downy mildew of leaf).
• Petriplates were placed in a growth chamber at 20°C and 100% RH for 24 h in
the dark, then under a 16 h light and 8 h dark photoperiod and 70% RH for 6
days.
• After 6 days sporangia were fully developed and isolate sporangia with the help
of distilled water and take the reading with haemocytometer.
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32. Contd:-
Treatments:-
• Grapevine plants were treated with 30 mM thiamine or water (control) on both
upper and lower leaf surfaces until the point of run-off using a compressed air
handsprayer device.
• Treated plants were kept in a growth chamber at 25°C, a 16 h light and 8 h dark
photoperiod, and 70% RH.
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33. Contd:-
Determination of disease incidence:-
• Grapevine leaf were kept in the growth chamber under controlled conditions
at 24°C, 16 h light and 8 h dark photoperiod, and 70% RH.
• Plants were incubated overnight in darkness at 80% RH and 20°C to allow
downy mildew sporulation and disease incidence was assessed as proportion
of plants showing necrosis, oil spots, and sporulation symptoms.
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34. Results:-
• Thiamine treatment elicited the expression of Phenylpropanoid pathway genes
which produce the stilbenes, phenolic compounds, flavonoids and lignin in the
grapevine plants.
• The total anti-oxidant potential of thiamine-treated plants was 3.5times
higher than the untreated-control plants.
• Molecular investigations have demonstrated that thiamine upregulated the
expression of CHS1 gene, which is responsible of flavonoid biosynthesis in
grapevine. Therefore, they investigated whether the activation of this gene by
thiamine correlated with an accumulation of flavonoids.
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35. Conclusions:-
• This work is the first to show the role of thiamine, as a vitamin, in the
modulation of grapevine plant secondary metabolism contributing to an
enhanced resistance to P. viticola, the most destructive fungal disease in
vineyards.
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36. Seminar Conclusions:-
• The ultimate conclusion of seminar is that Phenylpropanoid pathway produces
various kinds of secondary metabolite like flavonides and isoflavones and
flavones which protect the crop plant from the various pathogen attack and
UV radiation.
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37. References:-
• Dao, T. T. H., Linthorst, H. J. M., Verpoorte, R., Chalcone synthase and its functions in plant
resistance Phytochem Rev (2011) 10:397–412
• ANDERS, B. C., GREGERSEN,P. L., SCHRODER,J. AND COLLINGE, D. B., 1998, A chalcone
synthase with an unusual substrate preference is expressed in barley leaves in response to UV light
and pathogen attack. Pl. Mol. Biol.37: 849–857.
• BOUBAKRI, H., POUTARAUD, A., ALI, M. W., CLAYEUX, C. AND BALTENWECK, R. G.,
2013, Thiamine modulates metabolism of the phenylpropanoid pathway leading to enhanced
resistance to Plasmopara viticola in grapevine. BMC Pl. Biol.13: 1-15.
• ZABALA, G., ZOU, J., TUTEJA, J., GONZALEZ, D. O., CLOUGH, S. J. AND VODKIN, L. O.,
2006, Transcriptome changes in the phenylpropanoid pathway of Glycine max in response to
Pseudomonas syringae infection. BMC Pl. Biol. 6: 1-18.
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