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Elicitor-Mediated Enhancement of Folates in Coriander (Coriandrum sativum L.) and Elucidation of Molecular Mechanisms

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Title Elicitor-Mediated Enhancement of Folates in
Coriander (Coriandrum sativum L.) and
Elucidation of Molecular Mechanisms
 
Creator Bijesh, P.
 
Subject 04 Vitamins
30 Spices/Condiments
 
Description Folates (vitamin B9) are important cofactors as one-carbon donors, with a much
faster turn-over rate than ATPs. Folates are needed for DNA methylation, synthesis of
thymine, amino acids (methionine, glycine and serine), purine, pantothenate and formyl
methionine-transfer RNAs. Since animals cannot synthesize folates de novo, they solely
depend on plants and bacteria. While most developed countries adopted fortification
programs to alleviate folate deficiency, this approach is seldom successful in developing
countries where fortified foods are un-popular. Green leafy vegetables are rich inexpensive
sources of folates and other nutrients, of which coriander is vastly consumed on daily basis
in tropics. Coriander foliage has high folate content of 196 μg/100 g FW. Therefore, the
present study analysed folates in different commercial cultivars, characterized elicited
folates and developed a method that enhance stability during post-harvest storage. The
study has also elucidated sub-cellular mechanisms involved in folate enhancement and
identified folate-stabilizing proteins.
The concentration of total folates in foliages of ten commercially cultivated varieties
(cv.) of coriander was evaluated by microtestplate assisted-microbiological assay using
Lactobacillus rhamnosus (ATCC 7469). Certified seeds grown under identical
greenhouse conditions displayed marked variations in total folates as well as biomass
yield, the highest folates (1577 μg/100 g DW) being in cv. GS4 Multicut (45–50 days,
marketable stage). Coriander foliage showed high sensitivity to the light intensity where
2650 lux resulted in the highest foliar-folate, whereas in the dark, the content of folate
reduced to 1257 μg/100 g DW in cv. GS4 Multicut.
Folate enhancement was analysed using coriander callus cultures (to rule out other
effects). Growth regulators (6-benzylaminopurine, kinetin and abscisic acid) marginally
enhanced folates after 6 h whereas elicitor (salicylic acid - SA) doubled folates. In vivo
foliar applications of SA (250 μM, 24 h) doubled folates (3112 μg/100 g DW), the content
being sensitive to diurnal rhythms. Postharvest stability in treated foliage was 10% higher
than in control when stored at 25 °C and 4 °C. The elicitation of folates (5-CH3-H4folate,
5- and 10-CHO-H4folates) was differential, where the first two compounds nearly doubled
and the third increased six fold post-elicitation, with all forms showing concomitant
increase in in vitro bio-accessibilities, particularly after microwave drying.
Elucidation of biochemical and molecular mechanisms involved in elicitor-mediated
folate enhancement was done using coriander as well as the model plant Arabidopsis, due
to the availability of complete genomic data of the latter. In both plants, elicitation
decreased reactive oxygen species (ROS) with concomitant increase in folates and higher
activities of antioxidant enzymes (CAT, POX and SOD). Based on the screening of the
microarray data (GENEVESTIGATOR) of SA-response of Arabidopsis, expression profiles
of 19 genes were analysed by qPCR, which showed the down regulation of folate
biosynthetic genes and the up-regulation of folate-stabilizing genes, particularly that of a
putative folate binding protein (FBP) (AtFBP1, At5G27830) as well as a cytoplasmic folate
polyglutamate synthase (AtFPGS3, At3G55630). The accumulation of FBP was confirmed
by affinity column purification of the foliar proteins. Computational analysis of FBPs
revealed that the predicted model of one of the proteins - the AtFBP1 protein showed a
strong binding affinity with folic acid, with a free energy change (ΔG) of −9.3 kcal/mol at
the predicted binding site. Heterologous expression of AtFBP1 in a yeast model, tagged
with an N-terminal 6His, showed its over-expression upon induction, confirmed by western
blot analysis using specific antibody. Transformed-yeast cells showed significant uptake
and accumulation of the fluorescent-tagged folate, but not the wild type. The gene coding
for this FBP appears to be an interesting target for metabolic engineering in plants for
enhancing the concentration of folates and their post-harvest stability.
 
Contributor Bhagyalakshmi, N.
 
Date 2014
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Language en
 
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Identifier http://ir.cftri.com/12163/1/Bijesh%20Thesis.pdf
Bijesh, P. (2014) Elicitor-Mediated Enhancement of Folates in Coriander (Coriandrum sativum L.) and Elucidation of Molecular Mechanisms. PhD thesis, University of Mysore.