Antisence Rna technology

RNAi AND ANTISENSE RNA
TECHNOLOGY IN PLANTS

INTRODUCTION
• Diseases are often connected to the insufficient or excess production of
certain proteins. If the production of these proteins is disrupted, many
diseases can be treated or cured.
• Antisense technology is a method that can disrupt protein production. It
may be used to design new therapeutics for diseases in whose pathology
the production of a specific protein plays a crucial role.
• Delaying the ripening process in fruit is of interest to producers because it
allows more time for shipment of fruit from the farmer’s fields to the
grocer’s shelf, and increases the shelf life of the fruit for consumers.
Although ripening makes fruit edible and flavorful, it also begins the
gradual decline of the fruit towards softening and rot, causing losses for
producers and consumers

• The development of fruit in many plants follows a two-
step process. First, after pollination, parts within the
flower of the plant expand and develop into a full-sized
fruit. Second, the full-sized fruit undergoes the process
called ripening, a complex set of molecular and
physiological changes in the fruit.
• The ripening process brings dramatic changes to the
fruit, softening of cell walls, production of color
compounds, and changes in sugar content, flavor and
aroma.
• In tomatoes and many other fruits, ripening begins
when the fruit produces a volatile compound called
ethylene.

• Post harvest protection is an area of prime focus . Exploring
technologies for decay control and toxin inhibition for fruits, vegetables
and stored commodities is very important.
• Decay control, shelf-life extension and inhibition of toxin production.
• Decay and human pathogen control for fruits, vegetables and stored
commodities for shelf-life extension and reduction in food poison incidence
is very important.
• Two of the major methods used for the post harvest protection is-
• 1) RNAi
• 2) antisence RNA technology

RNAi
• suppression of a gene corresponding double stranded RNA is called RNA
interference (RNAi), or post-transcriptional gene silencing (PTGS).
• RNAi (RNA interference) refers to the introduction of homologous double
stranded RNA (dsRNA) to specifically target a gene's product, resulting in
null or hypomorphic phenotypes.
 The most interesting aspects of RNAi are the following:
• dsRNA, rather than single-stranded antisense RNA, is the interfering agent
it is highly specific
• it is remarkably potent (only a few dsRNA molecules per cell are required
for effective interference)
• the interfering activity (and presumably the dsRNA) can cause interference
in cells and tissues far removed from the site of introduction

How does RNAi work ?
• Since the only RNA found in a cell should be single stranded,
the presence of double stranded RNA signals is an abnormality.
• The cell has a specific enzyme (called Dicer) that recognizes the
double stranded RNA and chops it up into small fragments
between 21-25 base pairs in length.
• These short RNA fragments (called small interfering RNA, or
siRNA) bind to the RNA-induced silencing complex (RISC).
• The RISC is activated when the siRNA unwinds and the
activated complex binds to the corresponding mRNA using the
antisense RNA.
• The RISC contains an enzyme to cleave the bound mRNA
(called Slicer in Drosophila) and therefore cause gene
suppression. Once the mRNA has been cleaved, it can no
longer be translated into functional protein
Figure -Mechanism of action of RNAi. Double stranded RNA is
introduced into a cell and gets chopped up by the enzyme
dicer to form siRNA. siRNA then binds to the RISC complex and
is unwound. The anitsense RNA complexed with RISC binds to
its corresponding mRNA which is the cleaved by the enzyme
slicer rendering it inactive.

APPLICATIONS OF RNAi
• Various applications of RNAi include-
 RNAi for disease and pathogen resistance
 RNAi for male sterility
 RNAi and plant functional genomics
 Engineering plant metabollic pathway through RNAi

Trait Target Gene Host Application
Enhanced
nutrient content
Lyc Tomato Increased concentration of lycopene
(carotenoid antioxidant)
DET1 Tomato Higher flavonoid and b-carotene contents
SBEII Wheat, Sweet potato, Maize Increased levels of amylose for glycemic
management and digestive health
FAD2 Canola, Peanut, Cotton Increased oleic acid content
SAD1 Cotton Increased stearic acid content
ZLKR/SDH Maize Lysine-fortified maize
Reduced alkaloid production CaMXMT1 Coffee Decaffeinated coffee
COR Opium poppy Production of non-narcotic alkaloid, instead
of morphine
CYP82E4 Tobacco Reduced levels of the carcinogen
nornicotine in cured leaves
Heavy metal
accumulation
ACR2 Arabidopsis Arsenic hyperaccumulation for
phytoremediation
Reduced polyphenol
production
s-cadinene synthase gene Cotton Lower gossypol levels in cottonseeds, for
safe consumption
Ethylene
sensitivity
LeETR4 Tomato Early ripening tomatoes
ACC oxidase gene Tomato Longer shelf life because of slow ripening
Reduced
allergenicity
Arah2 Peanut Allergen-free peanuts
Lolp1, Lolp2 Ryegrass Hypo-allergenic ryegrass
Reduced production of
lachrymatory factor
synthase
lachrymatory factor synthase
gene
Onion "Tearless" onion

CO-SUPRESSION
• First discovery in plants found more than a decade ago.
• Occurred in petunias.
• Researchers trying to deepen the purple color but were surprised by the
results.
• Petunias were variegated or completely white.
• This phenomenon was termed co-suppression (later termed as RNAi),
since both the expression of the existing gene (the initial purple colour),
and the introduced gene (to deepen the purple) were suppressed.
• Co-suppression has since been found in many other plant species and also
in fungi. It is now known that double stranded RNA is responsible for this
effect.

Figure- A variegated petunia. Upon injection of the gene responsible for
purple colouring in petunias, the flowers became variegated or white rather
than deeper purple as was expected.

ANTISENSE RNA TECHNOLOGY
• Powerful technology that permits controlled silencing of a specific gene.
• Formulated by Dr. Hal Weintranb and colleagues at the basic science division in early 1980s.
• Tool used for inhibition of gene expression.
• Antisense RNA can be produced, for example, by inverting the coding region of a gene with
respect to its promoter. The antisense RNA can hybridize with its corresponding mRNA, making it
double-stranded. The double-stranded mRNA no longer can be recognized by the protein-
synthesizing machinery (the ribosomes), and thus expression of this mRNA is suppressed. Also, in
many systems double-stranded mRNA is very unstable and is broken down quickly. Thus, one can
inactivate specific genes while not interfering with others.
• Theories on how inhibition works
• Exact mechanism by which translation is blocked is unknown.
• Several theories include-
 That the ds RNA prevents ribosomes from binding to the sense RNA and translating (kimball,
november 2002)
 The ds RNA cannot be transported from within the nucleus to the cytosol which is were the
translation occurs(tritton,1998)
 ds RNA is susceptible to endoribonucleases that would otherwise not effect ss RNA but degrade
ds RNA ( Kimball, november 2002)

MECHANISM
• It has recently been shown that ds RNA in the cytoplasm triggers as yet
poorly understood cascade of events leading to the suppression of the
transcription of the gene producing the specific mRNA involved in the
cytoplasmic RNA duplex.

Several possible sequence specific sites of antisense inhibition. Antisense oligodeoxynucleotides are
represented by black bars. Antisense oligodeoxynucleotides can interfere with (I) splicing, (II) transport of the nascent
mRNA from the nucleus to the cytoplasm, (III) binding of initiation factors, (IV) assembly of ribosomal subunits
at the start codon or (V) elongation of translation. Inhibition of capping and polyadenylation is also possible. (VI)
Antisense oligodeoxynucleotides that activate RNase H (e.g., oligonucleotides with phosphodiester and
phosphorothioate
backbones) can also inhibit gene expression by binding to their target mRNA, catalyzing the RNase H
cleavage of the mRNA into segments that are rapidly degraded by exonucleases

• The inhibition of gene expression by antisense molecules is believed to occur by
a combination of two mechanisms:
(a) ribonuclease H (RNase H) degradation of the RNA and
(b) steric hindrance of the processing of the RNA(which physically prevent or inhibit
the progression of splicing or the translational machinery)
(c) RNase H is a ubiquitous enzyme that hydrolyzes the RNA strand of an RNA/DNA
duplex. Oligonucleotide-assisted RNase H-dependent reduction of targeted RNA
expression can be quite efficient, reaching 80–95% down-regulation of protein
and mRNA expression. Furthermore, in contrast to the steric-blocker
oligonucleotides, RNase H-dependent oligonucleotides can inhibit protein
expression when targeted to virtually any region of the mRNA. Thus, whereas
most steric-blocker oligonucleotides are efficient only when targeted to the 5′-
or AUG initiation codon region, phosphorothioate oligonucleotides, e.g., can
inhibit protein expression when targeted to widely separated areas in the coding
region

• In order for an antisense oligonucleotide to down-regulate gene
expression, it must penetrate into the targeted cells. To date, the precise
mechanisms involved in oligonucleotide penetration are not clear.
• Uptake occurs through active transport, which in turn depends on
temperature , the structure and the concentration of the oligonucleotide ,
and the cell line.
• At the present time, it is believed that adsorptive endocytosis and fluid
phase pinocytosis are the major mechanisms of oligonucleotide
internalization, with the relative proportions of internalized material
depending on oligonucleotide concentration.
• At relatively low oligonucleotide concentration, it is likely that
internalization occurs via interaction with a membrane-bound receptor .
De Diesbach et al. have recently purified and partially characterized one of
these receptors. At relatively high oligonucleotide concentration, these
receptors are saturated, and the pinocytotic process assumes larger
importance.

APPLICATION OF ANTISENSE RNA
TECHNOLOGY
• The application of Antisense technology is seen most in plant genetic
engineering.
• 1) Slow Ripening Tomato: In Tomato in the later stages of ripening
polygalactouronase gene is switched on coding for polygalactouronase
enzyme which breaks down polygalactouronic acid component of cell
walls resulting in softening which results in spoilt tomato. Transgenic
tomatoes were prepared containing antisense construct of the gene PG
resulting in reduced expression of PG and slow ripening and fruit
softening, improving shelf life.
• 2) Inactivate ethylene synthesis
• 3) Modification of flower color in decorative plants

ACC SYNTHASE GENE
• ACC stands for 1-amino-cyclopropane-1-carboxylic acid synthase gene.
• The enzyme 1-aminocyclopropane-1-carboxyllic acid (ACC) synthase, normally
found in carnations, is responsible for the conversion of S-adenosylmethionine
to ACC, which is the immediate precursor of ethylene.
• ACC synthase is a cytosolic enzyme that catalyzes the first committed step in
ethylene biosynthesis in higher plants. It is a key regulatory enzyme that
catalyzes the production of the ethylene precursor ACC from AdoMet (S-
adenosylmethionine).
• However, this enzyme is difficult to purify because it is labile and present in
low abundance in plant tissues. The molecular cloning and functional
expression of ACC synthase in E. coli and yeasts have facilitated biochemical
and structural studies of this enzyme.
• ACC synthase cDNAs and genomic sequences have been cloned from
numerous plant species.
• Ti plasmid of Agrobacterium tumefaciens used to transform tomato tissue with
antisense DNA for ACC synthase or ACC oxidase. When the genes for the two
enzymes are expressed in the reverse direction in the cell, the resulting
"antisense transcript" binds to and inactivates the "sense transcript" produced
by the wild-type gene, effectively shutting down expression of the wild-type
gene (Hamilton et al. 1990; Oeller et al. 1991; Picton et al. 1993). After
transformation, intact plants can be regenerated in tissue culture

The genetic manipulation of fruit
ripening
• Tomato ripening comprises a complex sequence of
biochemical and physiological changes affecting fruit color,
flavor and texture. As with all developmental changes it is
precisely co-ordinated by differential gene expression.
Evidence for this was first detected by Rattanapanone et
al.,(1978) in the form of mRNA changes. There are at least
19 ripening related m-RNA sequences that accumulate
during tomato ripening which have been cloned (Slater et
al.,1985). Of these, four have been identified and encode
cell wall degrading enzyme polygalacturonase (PG)
(Grierson et al., 1986, Della penna et al., 1986), a
protienase inhibitor, and two enzymes required for
ethylene synthesis, ACC synthase (Vander Stracten et al.,
1990) and the ethylene forming enzyme (ACC oxidase).

Ethylene Biosynthesis
• Ethylene is a gaseous
effecter with a very
simple structure.
ethylene has features
that identify it as a
hormone such as the
fact that it is effective at
nonomolar
concentrations.
• Biosynthesis of ethylene
in plants

• In higher plants, ehtylene is produced from L-
methionine. Methionine is activated by ATP to form
S-adenosylmethionine through the catalytic activity
of S-adenosylmethionine synthetase. Starting from
S-adenosylmethionine two specific steps result in
the formation of ethylene. The first step produces
the nonprotein amino acid 1-aminocyclopropane-1-
carboxylic acid (ACC). It is catalysed by ACC synthase
with pyridoxal phospate acting as a co-factor.
Formation of ACC is the rate limiting step in ethylene
biosynthesis. ACC synthase is encoded by a
medium-size multigene family. Various signals,
which influence ethylene synthesis result in
increased expression of single members of the ACC
synthase gene family. Production of ethylene from
ACC is catalysed by ACC oxidase.

• The concentration of ethylene in a plant
tissue is dependent on the rate of
biosynthesis and on diffusion of the gas.
Ethylene is neither actively transported nor
degraded.

Inhibiting ethylene synthesis
by gene silencing in
transgenic tomato plants • From the above functions of
ethylene, it is evident that if the
ethylene synthesis is hampered
using antisense RNA technology,
delayed fruit ripening could be
achieved. cDNAs for both ACC
synthase and ACC oxidase have
been cloned from tomato. RNAs to
ACC synthase have been expressed
in tomato using the CaMV
(Cauliflower Mosaic Virus) 35s
promoter and the NOS or CaMV
35s 3’ terminators. In case of ACC
oxidase, slower rates of ripening
were observed along with reduced
production of ethylene.
Inhibiting ACC oxidase (John et
al1995;Picton et al1993)

• Altered ripening of low –ethylene melon by
silencing ACO gene
Over-ripe melon Antisense ACC Oxidase melon
HARVESTED 38 DAYS POST-POLLINATION STORED AT 25ºc FOR 10 DAYS (J-C Pech
Toulouse)

Functions of ethylene
• Ethylene promotes maturation and abscission of
fruits. Many climacteric fruits such as apple,
banana and tomato show a strong increase in
ethylene levels at the late green or breaker stage.
• High amounts of ethylene degrade chlorophyll
and produce other pigment which imparts the
typical color to the mature fruit peel.
• Starch, organic acids and in some cases, such as
avocado lipids, are mobilized and converted to
sugars.

• Pectins, the main component of the middle lamella are degraded, because
of which the fruit softens. These metabolic activities are accompanied by
a high respiration rate and consequently by high oxygen consumption.
• Ethylene regulates senescense and fading of flowers and abscission of
petals and leaves. In most cases, flower formation is inhibited by
ethylene. Pineapple (Bromeliaceae) is an exceptional in that ethylene
promotes flower formation.
• Ethylene has evolved as the central regulator of cell death programs in
plants. In roots and in stems (in some plants), lack of oxygen induces
formation of intercellular spaces, the so called aerenchyma. Low oxygen
conditions in waterlogged roots, for instance, result in lysogenous
aerenchyma formation through programmed death of cells in the cortex.
This process is controlled by ethylene, as in programmed death of
endosprem cells during cereal seed development.

The genetic manipulation of fruit
softening
• Fruit ripening, as mentioned earlier, is an active process that, in
climacteric fruit such as tomatoes, is characterised by a burst of
respitation (respiratory climacteric), ethylene production, softening
and changes to colour and flavor. The peak of ethylene production
is significant because ethylene is known to be the phytohormone
that triggers ripening in climacteric fruit. The colour change results
from the degradation of chlorophyll and the production of red
pigment, lycopene. Flavour change occurs as starch is broken down
and sugars accumulate. A large number of secondary products that
improve the smell and taste of the fruit are also produced. The
softening of the fruit is largely the result of the cell wall degrading
activity of the enzymes polygalacturonase (PG) and pectin
methylesterase (PME). The PG enzyme is synthesized de novo
during ripening and acts to break down the polygalacturonic acid
chains that form the pectin ‘glue’ of the middle lamella, which
‘sticks’ neighbouring cells together.

• Polygalacturonase [PG; poly(1,4 -D-galacturonide) glycanhydrolase; EC 3.2.1.15]
is expressed in tomato only during the ripening stage of fruit development.
• PG becomes abundant during ripening and has a major role in cell wall
degradation and fruit softening.
• Tomato plants were transformed to produce antisense RNA from a gene construct
containing the cauliflower mosaic virus 35S promoter and a full-length PG cDNA in
reverse orientation.
• The construct was integrated into the tomato genome by agrobacterium-mediated
transformation.
• The constitutive synthesis of PG antisense RNA in transgenic plants resulted in a
substantial reduction in the levels of PG in mRNA and enzymatic activity in
ripening fruit.
• The steady-state levels of PG antisense RNA in green fruit of transgenic plants
were lower than the levels of PG mRNA normally attained during ripening.
• However, analysis of transcription in isolated nuclei demonstrated that the
antisense RNA construct was transcribed at a higher rate than the tomato PG
gene. Analysis of fruit from transgenic plants demonstrated a reduction in PG
mRNA and enzymatic activity of 70-90%.

INDIAN CONTRIBUTION
• NIGPR (National institute of plant genome research) in feb 2010 has
developed a tomato by antisense technology which can last long upto 45
days.
• NIGPR scientist had silenced the expression of 2 important gene which are
responsible for loss in firmness and textures during ripening.
• 2 genes that were silenced were alpha-man and beta hex of glycosyl
hydrolase, a kind of enzyme that breaks the chemical bond holding a sugar
to either another sugar or some other molecule like protein.

LONGEVITY IN BANANA
• Banana is a staple food for more than 450 million people particularly in
developing countries. It is a climacteric fruit hence possible to extend
longevity using antisense inhibition of ethylene production.
• The characteristics of banana genes encoding ACC synthase and ACC
oxidase was identified by constructing cDNA library using mRNA of ripening
banana fruit.
• In one design, cloned cDNA insert for ACC oxidase referred as BACS, 1231
bp long (ORF) which encodes for 315 amino acid length enzyme.
• The amino acid sequence of this gene exhibit conserved domain of ACC
synthase gene and resembles most of tomato ACC synthase gene with 62%
homology.
• Differential expression of enzyme activity at different stages of fruit
development was studied in banana. The differential longevity shows that
ethylene produced during fruit ripening was mainly from the peel

RIPENING IN WATERMELON
• Watermelon is also a typical climacteric fruit and there is sharp increase in
ethylene control during ripening stage that is acceptable for eating.
• Melon is less attractive model plant for manipulation due to its extensive
chromosome map and lack of mutants.
• Melon transformation was carried out using cotyledon explants and were
co-cultivated with engineered binary vector of harbouring an antisense
construct of ACC oxidase and marker gene like nptII

PROBLEMS WITH ANTISENSE
TECHNOLOGY
• Antisense technology has not been perfected. It is still difficult to express
aRNA only in targeted tissues. Precision gene therapy using aRNA needs to
be improved because as it is right now, aRNA sometimes binds to mRNA
that is not its target.
• Difficult of getting into the body.
• Other major challenge is its inevitable toxic effects. Although antisense
technology is engineered to be very specific, it still can cause unintented
damage.

POST HARVEST PROTECTION OF CEREALS, MILLETS & PULSES
CEREALS:
• The post harvest section of cereals provides ideas and solutions for
improved drying, reduction of damage and wastage, improved recording,
through no product marketing advice.
Cereal storage:
• Improvement in pest management systems for farm and central storage
including the design and implementation of rodent control programes.
• Reduction in the use of synthetic insecticides and their replacement with
botanicals.
• Investigation of insect behaviour in relation to pest management especially
the use of insect phenomenas and selection of grain varieties according to
insect resistance.
• Seed storage and control of grain quality in international aid shipments.

MILLET:
• Millet, which cover a number of botanical families, are short-cycle cereal
grown primarily in semi-arid regions. They are usually harvested after the
rainy season, so that when they reach maturity there is less danger from
humidity than from birds and other field pests, whether rodents or wild or
domesticated animals.
• Losses can be serious, depending on how long there is before harvesting
and how long harvesting itself takes. They can also be considerable during
transport, still often carried out manually. Losses can therefore be heavy,
even before the harvest is brought in and stored or sold.
Millet storage:
• Storing millet are done in farm or village granaries, traders' storehouses and
public or private storehouses, warehouses or silos.
• Traditional techniques that are commonly used include decorticating,
malting, fermentation, roasting, flaking and grinding. These methods are
mostly labour intensive and give a poor-quality product. Millets would
probably be more widely used if processing were improved and if sufficient
good-quality flour were made available to meet the demand.

Storage of millet in farm and village granaries

PULSES:
• Pulses are part of the staple diet of many countries and have the
advantage of adding some protein to diets based essentially on cereals,
but are harder to harvest and conserve than cereals.
• When they are mature, the dehiscent pods can open or burst, so that
many seeds fall to the ground; this happens during harvesting, but even
more so during transport.
• They are also more vulnerable to insect attack, particularly from weevils,
which lay their eggs on the pods or seeds before they are gathered.
• Pulses can have a high moisture content at the time of harvesting.
Effective drying is therefore important and this is generally done by
exposure to the sun on terraces or in openwork containers.

Pulses storage:
• Storing pulse grains after their harvest has always been a problem for
farmers as the stored grains are found to be most often infested with
pulse beetles.
• Freshly threshed pulse grains should be dried in the sun for 3-5 days. After
which it should be cooled and stored in suitable metal, plastic bins, large
earthen pots, brick, and cement storage structure with tight lids. While
storing the grains in the containers, farmers can apply a thick layer of
about 3 cm of locally available sieved sand (without any soil particles) on
top of the grains and tightly close the lid.
Effective control: Freshly threshed pulse grains
should be dried in the sun for 3-5 days.

• Future of RNA silencing technologies and its challenges in plants
• Can miRNAs Be Silenced Themselves?
• An alternative miRNA-like strategy was recently employed in plants, not to
artificially overexpress a particular miRNA but to antagonize an endogenous
miRNA's ability to cleave its specific target(s), providing a new functional
analysis tool to study plant miRNAs. This strategy, termed "target mimicry,"
relies on the expression of a small non-protein-coding mRNA that contains
a complementary miRNA binding site within its sequence.
• Can Promoter-Induced Silencing Be Used to Achieve Efficient Gene
Silencing in Plants?
• TGS accompanied by de novo methylation of a target promoter in plants
can be triggered by recombinant viruses or long hpRNA constructs
containing promoter sequences (Jones et al., 1998; Mette et al., 2000).
Such dsRNA-induced promoter silencing has long been proposed as a
potential technology for achieving potent and heritable gene silencing in
plants

• The Role of RNA Silencing in Plant Defense against Nonviral Pathogens:
Can This Be Exploited to Generate Resistance against a Broad Range of
Pathogens in Plants?
• viruses are a direct target of RNA silencing mechanisms, and hpRNA-based
constructs targeting viral RNAs have proven superior to previous
transgenic approaches for generating resistance in plants against viruses.
With the exception of Agrobacterium, whose T-DNA-encoded genes have
been shown to be targeted by PTGS, there has been no evidence that
genes of nonviral pathogens are a direct target of RNA silencing in plants.
Despite the aforementioned resistance to Agrobacterium, nematode, or
insects that has been achieved using hpRNA constructs in plants, it
remains to be seen whether direct targeting of pathogen-encoded genes
will become a practical approach for controlling a broad range of plant
diseases.

CONCLUSION
• Antisense technology is a formidable tool for investigating physiologic and pathologic
processes. In addition, it is soon likely to become a mainstay of therapy, particularly in
infectious diseases.
• Antisense technology is a powerful procedure that permits the controlled silencing of a
specific gene for investigations of mRNA and protein function
• Although there is still much to learn about the molecular processes and biological roles
of RNA silencing in plants, our current understanding of this RNA-mediated gene
control mechanism has already provided new platforms for developing molecular tools
for gene function studies and crop improvements.
• With RNAi, it would be possible to target multiple genes for silencing using a
thoroughly-designed single transformation construct. Moreover, RNAi can also provide
broad-spectrum resistance against pathogens with high degree of variability, like
viruses9. Recent studies have hinted possible roles of RNAi-related processes in plant
stress adaptation.
• Although much progress has been made on the field of RNAi over the past few years,
the full potential of RNAi for crop improvement remains to be realized. The
complexities of RNAi pathway, the molecular machineries, and how it relates to plant
development are still to be elucidated.

Antisence Rna technology

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