Its about how fruit ripening occurs and how we can manipulate ripening process by using biotechnology to delay ripening and to reduce postharvest losses
2. What is Fruit Ripening?
Highly coordinated
Genetically programmed
Irreversible phenomenon
Physiological, biochemical changes
Development of a soft and edible ripe fruit
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
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
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
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
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
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
-unmasking of previously present pigments by degradation of chlorophyll and dismantling of the photosynthetic apparatus
-volatile compounds such as ocimene and myrcene
-carotenoids such as β-carotene,xanthophyll esters, xanthophylls, and lycopene
-taste development increased gluconeogenesis, hydrolysis of polysaccharides, especially starch, decreased acidity, and accumulation of sugars and organic acids resulting in an excellent sugar/acid blend
-major textural changes Alteration of cell structure involves changes in cell wall thickness, permeability of plasma membrane, hydration of cell wall, decrease in the structural integrity, and increase in intracellular spaces
the activation of a high number of primary and secondary metabolic pathways that all
contribute to the overall sensory and nutritional quality of the fruit. This process involves the
expression of ripening-related genes that encode enzymes (proteins) involved in the various
ripening pathways (e.g., softening, color development). The whole process is under the control
of hormonal and environmental signals, amongst which ethylene plays a major role
Climacteric fruits
-ripening-associated increase in respiration and in ethylene production
-harvested at full maturity
-can be ripened off the parent plant
-The respiration rate and ethylene formation minimal at maturity
-raise dramatically to a climacteric peak, at the onset of ripening, after which it declines
Non-climacteric
-lack of ethylene-associated respiratory peak
-Can not undergo ripening process when detached from the parent plant.
-a very small quantity of endogenous ethylene
-do not respond to external ethylene treatment
-Show low profile and a gradual decline in their respiration pattern and ethylene production, throughout the ripening process
System 1 corresponds to low ethylene production in the pre-climacteric period of climacteric fruit, and is present throughout the development of non-climacteric fruit. System 2 refers to an auto-stimulated massive ethylene production called “autocatalytic synthesis”, and is specific to climacteric fruit.
System 1 refers to preclimacteric ethylene production, and System 2 to climacteric autocatalytic ethylene production. LeACS, Lycopersicon esculentum ACC synthase; LeACO, Lycopersicon esculentum ACC oxidase;
LeETR and NR, ethylene receptors. Eth+ and Eth– refer to the stimulation and repression, respectively of gene or protein expression
While LeACO1 and LeACO4 genes
are up-regulated at the onset of ripening, and continue being active throughout
ripening, LeACO3 displays only transient activation at the breaker stage of fruit
ripening (Fig. 16.2). It was shown that Le ACS6 and LeACS1A are expressed at the
pre-climacteric stage (system 1), while at the transition to ripening, LeACS4 and
LeACS1A are the most active genes (Fig. 16.2). Subsequently, LeACS4 continues to
express highly during climacteric phase, whereas the expression of LeACS1A
declines. The rise in ripening-associated ethylene production results in the induction
of LeACS2, and the inhibition of Le ACS6 and LeACS1A expression. This fine
tuning of the ACS genes is thought to be critical for the switch from pre-climacteric
system 1 to climacteric system 2
Recent studies demonstrated that the ethylene receptors are rapidly
degraded during fruit ripening, while the transcription rate remains high, and that
the receptor level determines the timing of ripening
ethylene-insensitive mutant ETR1 the ethylene receptor
Loss of firmness during heat treatment of acid fruit has been
attributed to acid hydrolysis of glycosidic bonds in cell wall
polysaccharides
The PG gene was the first to be cloned from tomato for studying textural regulation in ripening fruit and the transformed tomato withPGantisense gene resulted in improved fruit with firmer texture and an extended shelf life
Tomato germplasm altered in ripening. The dashed line separates mutants for which the corresponding gene has been cloned (1st tier) from those that have not (2nd tier). The third tier indicates transgenic lines altered in ethylene signaling
Never-ripe (Nr), which bears a dominant mutation that affects the ethylene response, and results in fruit producing
reduced amounts of ethylene and retaining very low ethylene responsiveness
Green-ripe (Gr) mutant corresponds also to a dominant ripening mutation lying
in a gene encoding a new component of ethylene signaling
ripening-inhibitor (rin) mutation is a recessive mutation that blocks the ripening process, and prevents ethylene production and responsiveness. The rin mutation encodes a MADS box-type transcription factor that is present in both climacteric and nonclimacteric fruit
The amount of ethylene produced can be controlled primarily by “switching off” or decreasing the production of ethylene in the fruit and there are several ways to do this. They include:
T-DNA map of RNAi-ACS binary vector. Antisense chimera was designed to be 50 bp shorter than the sense chimera, such that after transcription the antisense RNA
folds back and complements with sense RNA to form dsRNA molecules with loop in between.
Semi-quantitative RT-PCR analysis of ACS transcript levels in WT
and RNAi-ACS tomato lines at different stages of fruit ripening
introduction of truncated gene
constructs in the sense orientation, can result in
suppression of homologous host genes, a phenomenon called co-suppression
Papaya transgenic fruits (lines 5 and 12) of the same age as control fruits were collected, weighted daily and placed in closed containers. After 60 min a 1 ml sample was removed from the headspace and analyzed by gas chromatography.The fruits were then removed from the container and incubated at 25°C. The procedure was repeated for 7 days. Each point represents the average of ethylene produced by three fruits
Farmers can now wait for the fruits and vegetables to attain full maturity before they are plucked from their vines thereby as their produce can now be transported for longer periods of time, some of which would not even require refrigeration Extension in shelf life as fruits or vegetables as they stay fresher and nutritious for longer periods