Its about how fruit ripening occurs and how we can manipulate ripening process by using biotechnology to delay ripening and to reduce postharvest losses

1. Transgenics For Delayed Fruit Ripening
By
Sukanya 1385

2. What is Fruit Ripening?
Highly coordinated
Genetically programmed
Irreversible phenomenon
Physiological, biochemical changes
Development of a soft and edible ripe fruit

3. The molecular mechanisms controlling the ripening of fruit
Open Archive TOULOUSE Archive Ouverte

4. What are the changes?
Increased respiration
Chlorophyll degradation
Biosynthesis of carotenoids, anthocyanins, essential
oils, Flavor and aroma Components
Increased activity of cell wall-degrading enzymes
Transient increase in ethylene production

5. Major Developmental Changes during Tomato Fruit
Development and Ripening
The Plant Cell, Vol. 16, S170–S180 2004

6. Based on their respiratory pattern and ethylene biosynthesis
during ripening
V.Prasanna et al
Classification of fruits

7. Pathway for ethylene biosynthesis
Rate limiting step
Critical Reviews in Food Science and Nutrition, 47:1–19 ,2007

8. The expression of
ethylene biosynthesis
and ethylene
perception genes
during the transition to
climacteric in tomato
Kevany et al. 2007

9. Ethylene Perception and Signal Transduction
Bleecker and Kende, 2000

10. Structural components of fruits
Fruit pulp or the mesocarp
parenchymatus cells
complex network of polysaccharides and proteins
Plant polysaccharides play a major role in
storage, mobilization of energy and in
maintaining cell and tissue integrity due to
their structural and water binding capacity.
The primary cell wall contains
35% pectin
25% cellulose
20% hemicellulose
10% structural, hydroxyproline-rich protein

11. Enzymes Related to Pectin Dissolution
Critical Reviews in Food Science and Nutrition, 47:1–19 (2007)

12. Tomato: model systems for fruit development
and ripening
Globally cultivated fleshy fruit
World’s largest vegetable crop after potato
Indian production scenario-
3,50,000 hectares, 53,00,000 tons/year
Short generation time: 3-4 months
Simple genetics
Numerous characterized mutants
Cross fertile wild germplasm to promote genetic studies
Routine transformation technology
Postharvest losses-5 to 25% in developed countries
-20 to 50% in developing countries

13. Natural Mutants Affected in the Ripening Phenotype

14. Disadvantages of existing methods of storage
Labor intensive
costly
Occupies a large floor space
Poor heat transfer may occur resulting in poor product quality
Excessive dehydration in unpacked products
Chemical changes during freezing
-enzyme-activated browning
-development of rancid oxidative flavors
Textural changes during freezing
-mushy and watery

15. The use of 1-methylcyclopropene (1-MCP) on fruits and
vegetables
Inhibitor of ethylene perception
Easily released as a gas when the powder is dissolved in water
Approved by the Environmental Protection Agency (EPA) in 1999
Marketed as EthylBloc® by Floralife, Inc. (Walterboro, SC),AgroFresh.Inc., a
subsidiary of Rohm and Haas (Springhouse, PA)
C.B. Watkins,Biotechnology Advances 24 (2006) 389–409

16. Transgenic approach
Delayed
fruit
ripening
BLOCKING THE
PERCEPTION
OF ETHYLENE
BLOCKING THE
EXPRESSION
OF GENES
INDUCED BY
ETHYLENE
BLOCKING
ETHYLENE
SYNTHESIS

17. Regulation of Ethylene Production
a. Suppression of ACC synthase gene expression.
ACC (1-aminocyclopropane-1-carboxylic acid) (ACS2)
conversion of S-adenosylmethionine (SAM) to ACC
the second to the last step in ethylene biosynthesis
an antisense (“mirror-image”) or truncated copy of the synthase gene
Oeller et al, 1991
Yao et al,1999
Nath et al 2006

18. Antisense Technology
http://agbiosafety.unl.edu/flash/antisense.swf Journal of Plant Physiology.170,987– 995,2013

19. Null Mutation of the MdACS3 Gene
Apple cultivars homozygous or heterozygous for null allelotype
showed no or very low expression of ripening-related genes and
maintained fruit firmness
Aide Wang 2009

20. RNAi-mediated silencing
Chimeric RNAi-ACS construct designed to target ACS
homologs
Delayed ripening and extended shelf life for ∼45 days
Aarti Gupta, Ram Krishna Pal, Delayed ripening and improved fruit processing quality in tomato by RNAi-mediated silencing of three
homologs of 1-aminopropane-1-carboxylate synthase gene ,Journal of Plant Physiology 170 (2013) 987– 995

21. Regulation of Ethylene Production
b. Suppression of ACC oxidase gene expression.
It catalyzes the oxidation of ACC to ethylene
The last step in the ethylene biosynthetic pathway
Down regulation through anti-sense technology
Hamilton et al. 1990
Ye et al. 1996
Xiong et al. 2003

22. Ripening in papaya fruit is altered by ACC
oxidase cosuppression
Fig1:Map of the construct pKYCPACOO-1 containing the ACC oxidase
fragment cloned in PKYLX80 in the sense orientation. The ACC oxidase
fragment is flanked by the CaMV 35S promoter and the RUBISCO
terminator
Fig2: Ethylene production in papaya transgenic fruits.
Rodolfo Lo´pez-Go´mezet al.Transgenic Res. 18:89–97 2009

23. Regulation of Ethylene Production
c. Insertion of the ACC deaminase gene.
The gene is obtained from Pseudomonas chlororaphis
(a common nonpathogenic soil bacterium)
It converts ACC to a different compound
Reduce the amount of ACC available for ethylene production
90-97% reduced ethylene production
Klee et al.1991

24. Regulation of Ethylene Production
Plants transformed with ACC
deaminase
No differences in softness
Major difference in degradation
of fruit that occurs following
ripening
Klee et al. Ripening Physiology of Fruit from Transgenic Tomato (Lycopersicon
esculentum) Plants with Reduced Ethylene Synthesis Plant Physiol. Vol. 102, 1993

25. Regulation of Ethylene Production
d. Insertion of the SAM hydrolase gene.
The gene is obtained from E. coli T3 bacteriophage
SAM is converted to homoserine
The amount of its precursor metabolite is reduced
Matto, 2002
Good et al, 1994

26. Regulation of Ethylene Production

27. Regulation of Cell wall degradation
a.Polygalacturonase (PG)
degrades pectin
Antisense RNA techniques
The transgenic fruit with decreased levels of PG activity:
1)Do not get overly soft when ripe,
2)Show less damage due to fungal infection and
3)Have elevated levels of soluble solids
Bird et al, 1988

28. Regulation of Cell wall degradation
Chimaeric polygalacturonase (PG)
gene
Produce a truncated PG transcript
constitutively
Expression of the endogenous PG
gene was inhibited
C.J.S. Smith et al. Expression of a truncated tomato polygalacturonase gene inhibits
expression of the endogenous gene in transgenic plants Mol Gen Genet,224:477-481,
1999

29. Regulation of Cell wall degradation
b.Pectin methylesterase (PME)
Involved in metabolism of pectin
Break large polymers into shorter molecules
Antisense RNA approach
Transgenic fruit resulted in reduced pectin depolymerization
However there was no effect on firmness during ripening
Tieman et al,1992
Hall et al,1993

30. Regulation of Cell wall degradation
c.β-galactosidase
Normally upregulated during the early stages of ripening
Serves to remove pectic galactan side chains
Antisense regulation
d.Phospholipase D
Hydrolyze phospholipids
An antisense phospholipase D (PLD) cDNA Construct
resulted in a 30-40% reduction of PLD activity in ripe fruits
Transgenic fruits were firmer, possessed better red colour, and flavour
Pinhero et al. 2003
e.Deoxyhypusine synthase
Antisense gen copy of Senescence-induced deoxyhypusine synthase and
senescence-induced elf-5a
Pleiotropic effects on growth and development of tomato
Transgenics ripened normally, but exhibited delayed postharvest softening
Wang et al. 2005

31. Control of Ethylene Perception and signaling
Modifying ethylene receptors
The gene ETR1 encode an ethylene binding protein
Modified ETR1 lack the ability to respond to ethylene
Down-regulate specific tomato ethylene receptor isoforms using antisense
suppression have been reported for SlETR1, NR and SlETR4
Reporter genes related to ethylene responses and fruit ripening, LeCTR1 and
SlEILs genes, were also successfully silenced.
Fu et al, 2005
Zhu et al, 2006

32. Control of Ethylene Perception and signaling
Lucille Alexander Journal of Experimental J.C. Stearns, B.R. Glick ,Biotechnology Advances 21 (2003) 193–210 Botany, Vol. 53, No. 377

33. Anthocyanins Double the Shelf Life of
Tomatoes by Delaying Over ripening

34. Fruit specific and ripening related
promoters/cis-elements
Binding of specific trans-acting factors to the cognate cis-elements
Governs the spatial and temporal expression of a number of inducible genes
Tomato
E8 (Deikman et al., 1998)
2A11 (Vanand Houck, 1993)
Apple
ACO (Atkinson et al.,1998)
Melon
cucumisin (Yamagata et al., 2002)
WSP (Wu et al., 2003)
Strawberry
GalUR (Agius et al.,2005)
Grape
VvAlb1 (Li and Gray, 2005)
Banana
MaExp1 (Trivedi and Nath, 2004)
Research in Environment and Life Sciences, 2008

35. Advantages of Delayed fruit ripening
Assurance of top quality
Allowing the fruits to exude full quality
Consumers will get value for their money
Widening of market opportunities
Reduction in postharvest losses
http://www.isaaa.org/kc

36. References
James J. Giovannoni, Genetic Regulation of Fruit Development and Ripening, The
Plant Cell, Vol. 16, S170–S180, 2004
Antonio J Matas et al, Biology and genetic engineering of fruit maturation for enhanced
quality and shelf-life, Current Opinion in Biotechnology, 20:197–203, 2009
V. Prasanna et al, Fruit Ripening Phenomena–An Overview, Critical Reviews in Food
Science and Nutrition, 47:1–19 ,2007
M. Bouzayen et al, Mechanism of Fruit Ripening, Open Archive TOULOUSE Archive
Ouverte Eprints ID : 4525
Aide Wang et al, Null Mutation of the MdACS3 Gene, Coding for a Ripening-Specific
1-Aminocyclopropane-1-Carboxylate Synthase, Leads to Long Shelf Life in Apple Fruit,
Plant Physiology, Vol. 151, pp. 391–399, 2009
Rodolfo Lo´pez-Go´mez et al, Ripening in papaya fruit is altered by ACC oxidase
Cosuppression, Transgenic Res ,18:89–97, 2009
Aarti Gupta et al, Delayed ripening and improved fruit processing quality in tomato by
RNAi-mediated silencing of three homologs of 1-aminopropane-1-carboxylate
synthase gene, Journal of Plant Physiology 170,987– 995, 2013
Liu C et al, Cloning of 1-aminocyclopropane-1-carboxylate (ACC) synthetase cDNA
and the inhibition of fruit ripening by its antisense RNA in transgenic tomato plants, Chin
J Biotechnol. 1998;14(2):75-84
Gray J et al, Molecular biology of fruit ripening and its manipulation
with antisense genes, Plant Mol Biol. 1992 May;19(1):69-87

37. References
Oeller PW et al. Reversible inhibition of tomato fruit senescence by antisense RNA,
Science. 1991 Oct 18;254(5030):437-9
Harpster MH, Constitutive overexpression of a ripening-related pepper endo-1,4-beta-glucanase
in transgenic tomato fruit does not increase xyloglucan depolymerization or
fruit softening, Plant Mol Biol. 2002 Oct;50(3):357-69
Brummell DA et al. Cell wall metabolism in fruit softening and quality and its
manipulation in transgenic plants, Plant Mol Biol. 2001 Sep;47(1-2):311-40
Websites
http://agbiosafety.unl.edu/flash/antisense.swf
http://www.isaaa.org/kc
http://www.ukessays.com /essays/biology/quality-and-shelf-life-of-fruits-and-vegetables.
php
http://shodhganga.inflibnet.ac.in/bitstream/10603/4071/16/16_references.pdf
Books
Biology and biotechnology of the plant hormone ethylene
Edited by- A. Khanellis
Transgenic plants and crops
Edited by- M. Dekkerlne

38. THANK YOU