- The document describes a study that introduced the bacterial carotenoid biosynthetic genes CrtB and CrtI into wheat to increase its provitamin A content. - The study found that co-expression of CrtB and CrtI resulted in wheat lines with darker seed color and an approximately 8-fold increase in total carotenoid content and 76-fold increase in provitamin A content compared to unmodified wheat. - Analysis showed the transgenic lines had higher expression of endogenous carotenoid genes and dramatically elevated levels of beta-carotene and other provitamin A carotenoids in their seeds.

1. 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

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

3. What are carotenoids ?

4. What are carotenoids ?
• Photosynthetic pigments in chloroplast
• Coat by chlorophyll
• Ranging from red to yellow

5. 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

6. Carotenoids role..
• Antioxidant activity
• Antiradiant activity ( absorb light near blue
region at spectrum )
• Block free radicals from U.V ( Nutrailze T dimer )

7. Antioxidant activity
and free radicals

8. Sources of Vitamin A

9. Sources of Vitamin A

10. Symptoms of Vitamin A deficiency
• The most common cause of blindness in developing countries is
VAD*
• Night blindness and its worsened condition, xerophthalmia
a 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

11. 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.

14. Prevalence of vitamin A deficiency

15. Summary
• No carotenoids supplement –
• Problems with vision
• Problems with skin ( fast aging + radiation effect )

16. Why Triticum aestivum L ( Bobwhite )
• Bread Wheat Triticum aestivum L. make
94% of world consumption
• Vitamin A ( Retinol ) percent is Zero !

18. 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 ..

19. Transformation

20. 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 affected
as a result to CrtB , CrtI introduction ?

21. 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

22. • Carotenoid prolongs seed life

23. Goal of study
• Improve the amount of carotenoids in Wheat

24. What the benefits of this paper
• Valuable for breeding in wheat varieties
• Understanding mechanism regulating
carotenoid biosynthesis

25. 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

26. 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

27. • 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

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

31. Expected problems for CrtB , CrtI
introduction..
• Heterologous expression of carotenoid
biosynthetic genes
• Expression of unintended carotenoids
• Unexpected effect on the endogenous
carotenic genes in the transgenic plants

32. 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

33. Rubisco
• Rubisco .. Ribolose 1,5 biphosphate carboxylase /
oxygenase
• First step in carbon fixation .. Convert CO2
To energy rich molecules – Glucose -

34. Plasmids used

35. Plasmids construction
PU-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

36. 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

37. PAHC20 plasmid
• Selectable gene was inserted
• This gene resistance to herbicide phosphinothricin

38. 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

39. Transformation
• Transformed and grown in growth chamber
• Transformed plants will be selected by herbicide
• The regenerated plants then confirmed by PCR
using specific primers

40. • +ve PCR --> successful transgenic
• Self pollinated
• Analysed for carotenoid expression

41. 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

42. 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

43. Results
Generation of transgenic lines
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 !!

44. Expression of CrtB and/or CrtI changes the seed colour
of transgenic wheat
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

45. Expression levels of CrtI, CrtB determined by qPCR in
the endosperm of T3 Transgenic wheat lines
# 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-1
expression levels of CrtB in OEIB-2 and OEB-4 higher than those of CrtI in OEIB-2 and OEI-1
Due to higher expression driven by 1Dx5 promoter compared to 35S constitutive promoter

46. 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
Provitamin A dramatically increased among 3 lines elevated from 3.5 fold, 30
fold and 60 fold !! Suggesting that expresion of transgenes may include
additional carotenoid substrate in provitamin A pool of transgenic lines , up to
80% of carotenoids pool is provitamin A in trangenic compared to 9% of WT
•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

47. Average Carotenoids content of transgenic lines for each
carotenoid species
Increase in Provitamin A for transgenic lines
For Bobwhite.. Lutein was the major carotenoid

48. 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 pathway
Transgenic 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

49. 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 expression
In OEI-1 line ZDS gene slightly increased with downstream genes LCYB, LCYE upregulated
In OEB-4 and OEIB-2 most carotenoid biosynthetic genes was upregulated except HYD1
Expression of PSY1 and PSY2 was unaffected through transgenic lines
Moderate expression of LCYB and HDY2 in OEI-1 explained its carotenoid limited development in endosperm
Significant 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

50. 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 and
CrtI, Journal of Experimental Botany, Vol. 65, No. 9, pp. 2545–2556, 2014

51. 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