Using RNAi to improve plant nutritional value:-

from mechanism to application.

Presented by:- Ajay

kr.chandra

BT/02/09

Why , I have selected this topic

?‘Science’ (Couzin 2002) named RNAi as the “Technology of the year” , and‘Fortune’ Magazine has dubbed it as Biotech’s next “Billion Dollar Breakthrough” (Stipp 2003) in a cover story.

Historical aspects…• RNAi was first discovered in Petunia hybrida L. JORGENSONJORGENSON

(1990) by the introduction of chalcone synthase gene (CHS) in anthocyanin biosynthesis pathway.

Unexpectedly flower lost their clour and become colourless instead of purple ,but he was unable to explain.

later it is obtained that the silencing of endogenous homologous gene and this phenomenon was termed as “co-suppression”.

• RNAi occurrence is conserved among various organisms, also labeled as post-transcriptional gene silencing (PTGS) in plants, quelling in fungi (Romano and Macino 1992) and RNA interference in animals (Fire et al. 1998).

• Later on, Fire et al. (1998) elucidated the mechanism of RNAi in the nematode, Caenorhabditis elegans, and proposed the term “RNAi “.

• In 2006, Fire and Mello shared the

Nobel Prize in Physiology or Medicine for their work on RNA i in the nematode C. elegans.

Introduction • RNA interference (RNAi) is a sequence-specific gene silencing

mechanism, triggered by the introduction of dsRNA leading to mRNA degradation.

• It helps in switching the targeted gene on and off at transcriptional or, post-transcriptional level.

• RNAi can inhibit expression in 3 ways:- a) triggering destruction of mRNA in cytoplasm. b) inhibiting translation of mRNA. c) inducing chromatin modification within the promoter that

silence the genes. • In plants the RNAi technology has been employed successfully in

improvement to crop productivity and quality.

Components of RNAi• There are three main components , involved in RNA silencing.• these are as …. 1) small RNAs a) siRNAs b) mi RNAs 2) DICER 3) RISC

small RNAs• These involves mainly …. a) small interfering RNAs (siRNAs). = 21-25 bp dsRNA molecules. = exogenous in origin. = 100% complementary to target sequence. b) micro RNAs (miRNAs). = derived from ~70 bp ssRNA. = endogenous in origin. = forms a hairpin loop structure. = not 100% complementary to target sequence.

DICER• first discovered by Bernstein et al. (2001) in Drosophila .• It is a ribonuclease III type protein.• Commonly known as “fear of every dsRNA”.• It has 4 conserved domains , such as.. a) amino acid terminal helicase domain. b) duals RNase III motifs. c) dsRNA binding domain. d) PAZ (Piwi/Argonaute/Zwille) domain.

RISC• Stands for RNA induced silencing complex.• Large (~500kDa) ,RNA multi-protein complex ,which triggers mRNA

degradation in response to small RNAs.

• It has protein components like… = Argonaute family protein. = Helicase. = Ribonuclease • It performs… A) unbinding of dsRNAs (helicase activity) B) ribonuclease cleaves mRNAs (nuclease) C) amplification of silencing signal by RNA dependent RNA

polymerases. Finally , cleaved mRNA is degraded by exonucleases.

Mechanism

How it works?• Based on one gene – one enzyme hypothesis.

P1 P3P2 PP4sE4E1 E3E2 E5

RNAiGene-1 Gene-3Gene-4Gene-2 Gene-5

Nutritional improvements….

Flavr Savr • Transgenic for gene that slows ripening process .• Also “Rot-Resistant Tomato”.• It was produced by the Californian company Calgene.• The tomato was made more resistant to rotting by adding an antisense

gene which interferes with the production of the polygalacturonase(PG).

• The enzyme normally degrades pectin in the cell wall and results in the softening of fruit which makes them more susceptible to being damaged by fungal infections.

• Unmodified tomatoes are picked before fully ripened and are then artificially ripened using ethylene gas which acts as a phytohormone.

Tearless onion Dr Colin Eady, from The New Zealand Institute (CAFRI)

• Gluten (from Latin gluten , “glue") is a protein composite found in foods processed from wheat and related grain species, including barley ,rice and rye.

• Gluten gives elasticity to dough and helping to give chewy texture. • Gluten is the composite of a gliadin and a glutenin, which is

conjoined with starch in the endosperm of grains.

• Kusuba et al. (2003), by applying RNAi to improve rice plants with reduced level of glutenin and produced a rice variety called LGC-1 (low glutenin content 1).

• The low glutenin content was a relief to the kidney patients unable to digest glutenin.

• Certain allergies and neuropathies are also caused by gluten consumption.

Gluten free grains.

Increasing grain amylose content.• Foods rich in carbohydrates, such as fiber, are considered to be health promoting (Williams, 1995).

• Starch is a Homopolymer composed of D-glucoseD-glucose units held by alpha-glycosidic bondsalpha-glycosidic bonds.

• plant-derived carbohydrates is starch, which is composed of amylopectin and amylose as major nutritional source.

• High content of starch is found in cereals ,tubers, vegetables.

• Aiming to increase the relative content of amylose in wheat grains, a RNAi construct designed to silence the genes within seed-specific promoter in wheat ( Regina et al., 2006).

• This resulted in increased grain amylose content to over 70% of the total starch content (Tang et al., 2007).

-Carotene content in potato. -Carotene (pro-vitamin Apro-vitamin A) ,is necessary for vision ,proper growth of epithelial

cells.

• It is a carotenoids ,functions as AntioxidantsAntioxidants and reduce the risk of cancer initiated by free radicle.

• Increase in the content of health-promoting carotenoids, β-carotene and lutein, in potato which will mitigating the incidence of vitamin A deficiency in populations. (Eck et al. 2007).

• Increased consumption of this results into decreased incidence of Heart stroke ,skin Heart stroke ,skin and lungs cancerand lungs cancer.

• RNAi technology was used to enhance β-carotene content in potato by silencing the β-carotene hydroxylase gene , which converts β-carotene to zeaxanthin.

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

-Carotene pathway.

low caffeine content.• 10% of the coffee on the world market is shared by decaffeinate coffee

(DECAF). • A standard cup of filter coffee generally contains 60-150mg of caffeine,

while concentrations in decaf coffee are generally lower i.e 2-4 mg per cup.

• Its side effects include insomnia , restlessness insomnia , restlessness and palpitationspalpitations.• Caffeine is a (AlkaloidsAlkaloids) stimulant of the CNS, heart muscle and the

respiratory system, and has a diuretic effect.• Decaf is obtained from natural coffee by several ways: by using water or

solvent extraction. • RNAi technology has enabled the creation of varieties of Coffee that

produces natural coffee with low or very low caffeine content, thus by-pass the need of extraction (Van Uyen, 2006).

• caffeine content in coffee plants has been markedly reduced by RNAi-mediated suppression of the caffeine synthase gene.

Improving Essential Amino Acids in Crop Plants.

Improving edible oil quality .• Vegetable oils and fats constitute an important components in human diet.

• Rich source of energy and for certain vitamins, hormones and forms structural components of cells.

• Oil quality may be defined as the types and proportions of different fatty acids presents in the given oils.

• The unsaturated fatty acids ,body can’t synthesize and therefore , must be consumed in diet (Essential fatty acidsEssential fatty acids)

e.g. linoleic acid (18:2) Linolenic acid (18:3)

• Essential fatty acids are mostly called polyunsaturated fatty acids polyunsaturated fatty acids (PUFA).in fatty acid biosynthetic pathway, Acetyl CoA Acetyl CoA act as precursor for cholesterolcholesterol as well as palmitate.palmitate.

fatty acid pathway.

Lathyrism :- a case • In Ethiopia, Bangladesh and India, the people in the lower

socioeconomic class use a leafy vegetable known as Lathyrus sativus.

• It is a leguminous crop and contains a neurotoxin called β-oxalylaminoalanine-L-alanine (BOAA) .

• People consuming this vegetable suffer from a paralytic disease called, lathyrism.

• The disease paralyses people both temporarily and permanently. • RNAi technology can be used to silence the gene responsible for

production of BOAA, a anti-nutritional factor. • Bringing down the levels of BOAA to a safe concentration, rather

than totally silencing the concerned genes, may overcome this obstacle (Williams et al., 2004).

Edible Vaccines• Introduction of orally active antigenic proteinorally active antigenic protein.• Potato, banana, corn and tomato are targets.• Transgenic Plants Serving Human Health.• Viral proteins for infectious diseases such as - Diarrhea

- Hepatitis B - Measles

Advantage: Ability to efficiently vaccinate people world-wide.

Future with RNAi

Conclusions and perspective.• The nutritional value of human vegetable foods has an increasing role

in the prevention of various human diseases associated with malnutrition.

• RNAi triggered by dsRNA has great advantages over other approaches, owing to its higher gene silencing efficiency and the shorter time needed to screen the targeted plants.

• Tissue- or organ-specific RNAi vectors are needed to achieve targeted gene silencing in particular plant tissues and organs with minimal interference to the normal plant lifecycle.

• If judiciously used, this technology may go a long way to narrow the gap through production of disease-, insect- and virus resistant, nutritionally rich and toxic-free crops.

• Thus, Current agricultural technology needs more and more molecular tools to reduce current crop loss and feed extra mouths, which will increase by two billion over the next 30 years.

References… • Huang, J. et al. (2002) Enhancing the crops to feed the poor. Nature

418, 678–684.• Tang, G. et al. (2003) A biochemical framework for RNA silencing

in plants. Genes Dev. 17, 49–63. • G. Tang and G. Galili :-Using RNAi to improve plant nutritional

value: from mechanism to application; TRENDS in Biotechnology; Vol.22 ;9 September 2004.

• Brown ME, Funk CC (2008) Climate: food security under climate change. Science 319:580–581.

• Hirschi K (2008) Nutritional improvements in plants: time to bite on biofortified foods. Trends Plant Sci 13:459–463.

• www.researchgate.net• Singh B.D :- plant breeding, principles and methods ; VIII th edition ;

389-639.

Thanks