Hashemite University

Enrichment of provitamin A content in wheat ( Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic CrtB and CrtI

Course : Special Topics B

Instructor : Dr. Abdullatif Al Ghzawi

Presenter : Belal Abu hanieh

Enrichment of provitamin A content inwheat ( Triticum aestivum L.) byintroduction of the bacterial carotenoidbiosynthetic CrtB and CrtI

What are carotenoids ?

What are carotenoids ?

• Photosynthetic pigments in chloroplast

• Coat by chlorophyll

• Ranging from red to yellow

What are carotenoids ?

• Found mostly in many organisms But..

• Its biosynthetic enzymes may not found in some species

• Carotenoids which converted to Vitamin A called Pro-Vitamin A

Carotenoids role..

• Antioxidant activity

• Antiradiant activity ( absorb light near blue region at spectrum )

• Block free radicals from U.V ( Nutrailze T dimer )

Antioxidant activityand free radicals

Sources of Vitamin A

Sources of Vitamin A

Symptoms of Vitamin A deficiency

• The most common cause of blindness in developing countries is VAD*

• Night blindness and its worsened condition, xerophthalmiaa condition where the eyes fail to produce tears.

• Dead epithelial and microbial cells accumulate on the conjunctiva and form debris that can lead to infection and possibly blindness

• Can lead to impaired immune function, cancer, and birth defects

• Collections of keratin in the conjunctiva, known as Bitot's spots, are also seen

* VAD .. Vitamin A deficiency

Symptoms of Vitamin A deficiency

• Night blindness

• The difficulty for the eyes to adjust to dim light.

• Affected individuals are unable to distinguish images in low levels of illumination.

• People with night blindness have poor vision in the darkness, but see normally when adequate light is present.

Prevalence of vitamin A deficiency

Summary

• No carotenoids supplement –

• Problems with vision

• Problems with skin ( fast aging + radiation effect )

Why Triticum aestivum L ( Bobwhite )

• Bread Wheat Triticum aestivum L. make 94% of world consumption

• Vitamin A ( Retinol ) percent is Zero !

Why Crt B and Crt I ?

• But not other genes in the chain of the provitamin A biosynthetic pathway ?

• They are the most confirmed stable genes when introduced

• Other genes cause many problems when introduced

• Crt B and Crt I increase carotenoid content +vely ..

Transformation

Objectives..

• Was the introduction of CrtB , CrtI to Bobwhite cultivar successfully done ?

• How much fold Pro-Vitamin A affected ( increased )?

• Does the phenotype of endosperm pigment affectedas a result to CrtB , CrtI introduction ?

Rational..

• The enrichment of Pro-Vitamin A will make the wheat a balanced food

• Third world people will no long still depend on other food sources of Vitamin A not available

• Vitamin A is a protection of many diseases

• Carotenoid prolongs seed life

Goal of study

• Improve the amount of carotenoids in Wheat

What the benefits of this paper

• Valuable for breeding in wheat varieties

• Understanding mechanism regulating carotenoid biosynthesis

Other works on CrtI

• This group of researchers has performed a similar work in 2009 to enrich Provitamin A content by introduction of zea maize genes PSY1 and CrtI

• The amount was moderate

• The process was not clearly understood

Materials and methods> Carotenoid biosynthetic pathway..

• Many enzymes control the conversion of core material geranyl geranyl pyrophosphate to ( Pro-Vitamins )

• Example :

• CrtB.. Bacterial phytoene synthase

• CrtI.. Carotene desaturase

• In higher plants .. Carotenoids biosynthesis originates in the plastids and catalyzed by the nuclear-encoded enzymes

• These enzymes then .. Imported post transitionally into the organelle

We note that CrtB work as condesnser enzyme also we note that CrtI can do multiple functions by it self ( Desaturation+ Isomerization )

Expected problems for CrtB , CrtIintroduction..

• Heterologous expression of carotenoidbiosynthetic genes

• Expression of unintended carotenoids

• Unexpected effect on the endogenous carotenic genes in the transgenic plants

Plasmids

• Plasmids ( PACCRT-EB ) and ( PYPIET4 ) served as source of the coding sequence of CrtB , CrtI of Erwinia Uredevora

• In order to direct the expression of CrtB, CrtI in wheat plasmids

• Sequence encoding transit peptide ( TP ) of the small subunit of Rubisco ( rbc S ) from pea were fused with the CrtI, CrtB during Vector construction

Rubisco

• Rubisco .. Ribolose 1,5 biphosphate carboxylase / oxygenase

• First step in carbon fixation .. Convert CO2

To energy rich molecules – Glucose -

Plasmids used

Plasmids constructionPU-I plasmid ..

1. CaMV 35 s - TP– CrtI – Nos were cut by Hind III and EcoR I from PYPIET4

2. This fragment fused with PUC18 vector at Hind III and EcoR I sites

PTP- B plasmid

1. CrtB and TP was amplifed

2. Obtained fragments then spliced by overlap-PCR

3. The resulting Tp-CrtB fragment digested with SmaI, PstI

4. Ligated under control of 1Dx5 promoter

PAHC20 plasmid

• Selectable gene was inserted

• This gene resistance to herbicide phosphinothricin

Wheat transformation and regeneration

• Bobwhite cultivar was transformed with plasmids with the molar ratios

• PTP-B : PU-I : PAHC20 .. 2 : 2 : 1

• PTP-B : PAHC20 .. 3 : 1

• PU-I : PAHC20 .. 3 : 1

Transformation

• Transformed and grown in growth chamber

• Transformed plants will be selected by herbicide

• The regenerated plants then confirmed by PCR using specific primers

• +ve PCR --> successful transgenic

• Self pollinated

• Analysed for carotenoid expression

Analysis of carotenoid composition by HPLC

• Seed samples prepared to fine flour

• Quantitive analysis..

To quantify carotenoids and chlorophylls

3 leaves extaction from transgenic

HPLC

3 leaves extaction from untransformed

Quantitive analysis qRT-PCR

• RNA extracted from seeds of 25-30 day after pollination from 4-month old seedling of transgenic and untransformed plants

• Data analysed using lightcycler software and normalized to the expression β-actin gene

As the relative constitutive expression throughout the wheat developmental process

Results

Transformation ( CrtB , CrtI ) Herbicide selection Screen out by PCR

Self pollination of +ve PCR transgenic wheat

**Not all Regenerated plants are (+) positive PCR transgenic lines !!

Generation of transgenic lines

co-expression of both genes resulted in darker red/yellow grain phenotype,

the grain appearance more wrinkled may be due to reduction in grain weight

Expression of CrtB and/or CrtI changes the seed colourof transgenic wheat

# of Seed / spike not affected

Transgenic lines show loss in weight

Total carotenoid content increase of approximately 8-fold of seed dry weight

β-carotene increase of 65-fold of seed dry weight

provitamin A content (sum of α-carotene, β-carotene, and β-cryptoxanthin) increase of 76-fold of seed dry weight

expression levels of CrtB, CrtI in OEIB-2 slightly higher than the corresponding lines OEB-4 and OEI-1expression levels of CrtB in OEIB-2 and OEB-4 higher than those of CrtI in OEIB-2 and OEI-1

Expression levels of CrtI, CrtB determined by qPCR in the endosperm of T3 Transgenic wheat lines

Due to higher expression driven by 1Dx5 promoter compared to 35S constitutive promoter

Expression of CrtB or/and CrtI enhances carotenoids accumulation in seeds in transgenic wheat

For total carotenoid and provitamin A content amount compared to Bobwhite

•CrtI ( OEI-1 ) .. No significant total carotenoid significant provitamin A

•CrtB (OEB-4 ) .. significant difference total carotenoid and provitamin A suggested changes • in metabolic pathway•CrtB+CrtI ( OEIB ) .. significant difference total carotenoid and provitamin A

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Average Carotenoids content of transgenic lines for each carotenoid species

Increase in Provitamin A for transgenic lines

For Bobwhite.. Lutein was the major carotenoid

HPLC chromatogram of carotenoids extracted from grains of T3

Transgenic and control lines

In untransformed Bobwhite (E) .. Lutein was detected where other carotenoids was negligible suggesting a bias toward β, έ branch of metabolic pathwayTransgenic OEI-1, OEB-4 and OEIB-2 lines notably have increased β-carotene levels of 3.5 fold, 27 fold and 56 fold respectively other carotenoids unlike β-carotene increased moderately

Effect of CrtI and CrtB on endogenous carotenoid biosynthetic genes expression and on accumulation in wheat ( endosperm and leaf )

Expression of endogenous genes analyzed in both endosperms and leaves from transgenic and control wheat lines

VC-10 control line and Bobwhite show identical carotenoid expressionIn OEI-1 line ZDS gene slightly increased with downstream genes LCYB, LCYE upregulatedIn OEB-4 and OEIB-2 most carotenoid biosynthetic genes was upregulated except HYD1Expression of PSY1 and PSY2 was unaffected through transgenic linesModerate expression of LCYB and HDY2 in OEI-1 explained its carotenoid limited development in endospermSignificant increase expression of other carotenoids in OEB-4 and OEIB-2 ( PDS, ZDS, LCYE, LCYB, HYD2) was consistent with the drastic elevation of provitamin A

References

Cheng Wang, Jian Zeng, Yin L, Wei Hu, Ling Chen, Yingjie Miao, Pengyi Deng, Cuihong Yuan,Cheng Ma, Xi Chen, Mingli Zang, Qiong Wang, Kexiu Li, Junli Chang, Yuesheng Wang,Guangxiao Yang and Guangyuan He (2014). Enrichment of provitamin A content in wheat (Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic CrtB andCrtI, Journal of Experimental Botany, Vol. 65, No. 9, pp. 2545–2556, 2014

Supplementary material

http://nutritiondata.self.com/facts/cereal-grains-and-pasta/5744/2

http://www.cancer.org/treatment/treatmentsandsideeffects/complementaryandalternativem

edicine/herbsvitaminsandminerals/vitamin-a-and-beta-carotene

http://www.ncbi.nlm.nih.gov/pubmed/17541775

http://lipidlibrary.aocs.org/plantbio/carotenoids/index.htm

http://www.nutri-facts.org/#

http://www.ncbi.nlm.nih.gov/pubmed/21366698

http://www.nutri-facts.org/eng/topic-of-the-month/detail/backPid/53/article/the-role-of-

carotenoids-in-disease-prevention/

http://www.asknature.org/strategy/f21892e4fc7078700f1690880da5aec7