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WELCOME

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CHALCONE SYNTHASE AND ITS FUNCTION IN PLANT RESISTANCE

BY: AVINASH SHARMA

PALB-3235 Sr M.Sc.

Contents:-

• Introduction• Control of CHS Activity• CHS localization • Regulation of CHS Gene Expression• CHS activity in Plant Resistance• Case Study• Seminar Conclusions• References9/6/2014

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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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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Phenylpropanoid Metabolic Pathway:-

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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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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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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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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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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 controlB) Control of CHS turnoverC) Control of CHS through trans-genes

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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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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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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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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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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 loci9/6/2014

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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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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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.9/6/2014

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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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 graminisErysiphe graminisUV

Flavonoids

3. Glycine max Soybean Pseudomonas syringae pv glycinea, Phytophthora megaspermaf. sp. Glycinea.

Flavonoids

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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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Contd:-

Sl. No. Host Crop Pathogen/stresses Metabolites

9. Picea abies Norway spruce Ceratocystis polonica, Ophiostoma polonicum and wounding

Catechin

10. Arabidopsis thaliana Thale cressLow temperature, UV-B, UV-A, and blue Light

Anthocyanins

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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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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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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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CASE STUDY

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Department of Plant Biotechnology 289/6/2014

* 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

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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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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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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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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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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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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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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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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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: 1-18.9/6/2014

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