Gene expression modification for post-harvest improvement on horticultural crops

1. Gene expression modification for post-harvest
improvement on horticultural crops
By: FZ.amirmohammadi
May 2017
Post harvest
Ferdowsi university of Mashhad, Iran
Faculty of Agriculture Engineering

2. Introduction
 commercial advantages: tomatoes (do not ripen too quickly) melons ( do not spoil
soon after arriving) .
 Fruits are one of the major sources of vitamins, essential nutrients, antioxidants
and fibers in human diet.
 Short post-harvest shelf life and Senescence is one of the major horticultural
problems of many climacteric fruit (e.g. apple, avocado, banana, mango, peach,
pear etc.)
 greatly affects the consumer preference and export of fresh fruits.
 The main challenges to producers of fruit species continue to be how to arrest and
deliver to market the desirable color, taste, aroma and texture attributes of ripe
fruit and delaying the negative consequences of over-ripening.
 In recent years molecular biological approaches have been utilized to identify
genes that may be involved in the initiation and regulation of the senescence
program.
 The identification and characterization of ripening and senescence-related genes
has begun to provide us with an understanding of the process of senescence.
 senescence, a process that limits yield, nutritional value, and marketability of
many crops, will lead to ways of manipulating senescence for agricultural
applications.

3. System ISystem I,II
 Increased respiration.
 Chlorophyll
degradation.
 Biosynthesis of
carotenoids,
anthocyanins
 essential oils
 Flavor and aroma
Components
 Increased activity of cell
wall-degrading enzymes
What are this changes?
Classification of fruits: Based on
their respiratory pattern and
ethylene biosynthesis during
ripening.

4. •Regulation of Ripening and biosynthesis ethylene
control
O2,CO2,temperatuA,
MCA)
Avoiding
stress,Oxidaition
Chemical adsorbtion
Biosynthesis pathway
inhibition
Perception receptor
inhibition (1-mcp)
Environment control
Transgenic-induse
tolerance
(viruse-agrobacterium)
Insensetive mutant
Gene silencing
Antisense
Mirna
RNAi
control strategis
Genetic strategies
Chemical strategies
Ethylene control strategies Genetic
 Antisense gene silencing
 Virus-induced gene silencin
 Insensitive mutants
 Transgene-induced toleranceVentilation
 Biosynthesis pathway inhbition
 Reception receptor inhibition Ethylen
 Nanotechnology for ethylene control(Recent
results on the use of nanotechnology sensu
latofor cut flower vase life improvement)

5.  plant development, growth and
survival.
 seeds germination, flower and fruit
development,defense mechanisms,
 fruit ripening in climacteric fruits.
 regulates all physiological processes,
stress response biotic and abiotic.
 Very simple molecule
 A gas
 An important chemical feedstock
 A natural plant hormone
 Ethylene is biologically active at very low
concentrations of around 0.01 to 1.0 part per
million (ppm).

6. •SAM =S- Adenosylmethionine
ACC =Aminocyclopropane carboxylic acid system 1
• ACC synthase
(ACS)
SAM
• ACC oxidase
• (ACO) or (EFE)
•
ACC
• Oxidation
products
C2H2
• Ethylene
receptors
Signal
transduction
At the onset of fruit
ripening , expression
of multiple ACC
synthase genes are
activated
Mechanism of RipeningPathway of ethylene
Biosynthesis and Metabolism
Responses
Fruit ripening Defense signaled
geneexpression
SAM synthetase
Antisense ACO
CO supppression
ACO
Mu(etr1)Ov ACC deaminize.
Expression of bacterial .
Antisense ACSCH
3
MACC
*
*
*

7. Simplified ethylene pathway.
A:Basal production of ethylene in the flowers during development before senescence.

8. System 1 transition System2
SAM ACC C2H4 Perception
ACOACS
LeACS4
LeACS6
LeACS1A
LeACS2
Developmentally
regulated
Positive regulation –
steep increase in
ethylene production
Nr
ev+
rin
Model proposing the differential regulation
of ACS gene expression during the transition
from system 1 to system 2 ethylene
synthesis in tomato. The symbols ±ve
(negative) and +ve (positive) refer to the
action of ethylene on signalling pathways
resulting in repression (±ve) or stimulation
(+ve) of ACS gene expression.
eight ACS genes have been
identi®ed tomato(LEACS1A,
LEACS1B and LEACS2-7),
LEACS1A
auto-inhibitory
Negative feedback
auto-stimulatory
positive feedback
MdACS1*
silencing

9. • 1-down-regulation of ACO in otherflower species such as begonia and torenia
• down-regulation of the ACS gene in carnation also reduced ethylene production.
• Use of antisense sequences in petunia for ACO and ACS, derived heterologously from
broccoli, also delayed floral senescence
• 2-receptor inhibition
• Dianthus Campanula, Kalanchoe (Other genes in the ethylene signalling pathway such
as EIN2, whichis down-stream of the receptor, have also been down-regulated
inornamental species such as petunia resultingin delayed senescence.
•‫تبدیل‬ ‫از‬ ‫ممانعت‬ACC‫ژن‬ ‫بیان‬ ‫میزان‬ ‫کاهش‬ ‫با‬ ‫اتیلن‬ ‫به‬ACC‫منجر‬ ‫سنس‬ ‫آنتی‬ ‫تکنیک‬ ‫از‬ ‫استفاده‬ ‫اکسیدازبا‬‫به‬
‫شد‬ ‫میخک‬ ‫بریده‬ ‫شاخه‬ ‫گلهای‬ ‫ماندگاری‬ ‫افزایش‬.
•‫زن‬ ‫بیان‬ ‫کاهش‬ ‫طریق‬ ‫از‬ ‫گل‬ ‫های‬ ‫بافت‬ ‫در‬ ‫پیری‬ ‫های‬ ‫فرایند‬ ‫تاخیر‬EIN2‫در‬ ‫سنس‬ ‫آنتی‬ ‫تکنیک‬ ‫از‬ ‫استفاده‬ ‫با‬
‫شد‬ ‫گزارش‬ ‫اطلسی‬ ‫و‬ ‫توتون‬ ‫گیاهان‬.
• in cut flowers (Changes in gene expression)

10. Tomato is a good model system to investigate the mechanistic basis of fruit
ripening
diploid genetics, a range of well-characterized single gene mutants [available from the Tomato
Genomic Resource Center (TGRC)],
recombinant inbred lines (RILs; Eshed and Zamir, 1994),
and mapping populations and an excellent and well-annotated genome sequence (Tomato
Genome Consortium, 2012)
it is easily transformed, and mechanistic hypotheses can be tested using stable transgenic lines or
by virus-induced gene silencing (VIGS) .
Several databases are available for exploring genome and expressed sequence tag (EST)
sequences (Sol Genomics Network; Bombarely et al.,2011) and for gene expression analysis
(Tomato Expression Database; Fei et al., 2006)
Along with tomato, the sequencing of numerous fleshy fruit genomes including papaya (Ming et
al., 2008), strawberry (Shulaev et al., 2011), grape (Jaillon et al., 2007), apple
(Velasco et al., 2010), cucumber (Huang et al., 2009), cacao (Argout et al., 2011), banana
(D’Hont et al., 2012), melon (Garcia-Mas et al., 2012), kiwifruit (S. Huang et al., 2013)
pear (Wu et al., 2013), sweet orange (Q. Xu et al., 2013), watermelon (Y. Xu et al., 2013), and
pepper (Kim et al., 2014) has now provided the tools to reveal the underlying mechanisms
governing fruit development and ripening.

11. Several mutations that
affect ethylene
perception and
signaling interfere with
fruit ripening.
Receptor
CTR
Air or
Ethylene
Wild type
Green-ripe
Never-ripe
Never-ripe2
Ethylene perception mutants interfere with
ripening

12. Summary of ethylene synthesis and signaling
Ethylene Biosynthesis
SAM
ACC
C2H4
ACS
ACO
ETR1 and others
Ethylene Signaling CTR1
EIN2
EIN3, EILs
ERF1 and ERFs
ETP1 and ETP2
RTE/GR
EBF1 and EBF2
ethylene receptor
transcription factor
transductional cascade
ethylene responseive gene
LeETR1
LeETR2
NR
LeETR4
LeETR5
LeETR6
CTR1

TCTR2
 ER50

14. ETR1
histidine kinase receiverGAF
ethylene
binding
All ethylene receptors have a sensor domain that
can be subdivided into a transmembrane domain
and a GAF domain, a histidine kinasedomain and
a response domain. The binding of ethylene to the
receptor is mediated by a copper cofactor.CTR1, a
protein kinase with homology to the Raf family.
ERS2
NH2
There are three transmembrane segments
in the ethylene binding domain of ETR1
(four in subfamily II receptors)
What type of receptor is the ethylene
receptor?(negative regulate)
Arabidopsis
ethylene
receptor familyII
ERS1 ETR1
EIN4 ETR2
hydrophobic,
3alfahelix

15. The involvement of ethylene response factor (ERF) transcription factor (TF) in the
transcriptional regulation of ethylene biosynthesis genes during fruit ripening remains
largely unclear. In this study, 15 ERF genes, designated as MaERF1–MaERF15, were
isolated.

16. What does the ethylene-signaling pathway look like?
2
3
1
protolithic release transcriptional cascade
Activate the CTR1 protein kinase
4

18. Antisense RNA is a single-stranded RNA that is complementary
to a messenger RNA (mRNA) strand transcribed within a cell.
They are introduced in a cell to inhibit the translation machinery
by base pairing with the sense RNA and activating the RNase H,
to develop a particular novel transgenic.
mRNA sequence(sense) AUGAAACCCGUG
Antisense RNA UACUUUGGGCAC
What is antisense RNA??

19. Mechanism of antisense activity

20. The intended effect of the both technique is same but the
processing is a little bit different in both.
Antisense technology degrade the mRNA by RNaseH while RNAi
employed enzyme Dicer for degradation.
RNAi are twice larger than antisense oligonucleotide.
How it Differ from RNAi ??

21. Mechanism of RNA interference

22. Delayed ripening and improved fruit processing quality in
tomato by RNAi-mediated silencing of three homologs of 1-
aminopropane-1-carboxylate synthase gene

23. The phytohormone ethylene (ET) is a crucial signaling
molecule that induces the biosynthesis of shikonin
r, positive regulators
function of LeACS1, a key gene encoding the 1-
aminocyclopropane-1-carboxylic acid synthase for ET
biosynthesis in L. erythrorhizon hairy roots,
using overexpression and RNA interference (RNAi)
strategies.
The results showed that overexpression of LeACS-1
significantly increased endogenous ET concentration and
shikonin production, consistent with the up-regulated genes
involved in ET biosynthesis and transduction,
Transgenic analysis reveals LeACS-1 as a positive regulator of
ethylene-induced shikonin biosynthesis in Lithospermum
erythrorhizon hairy roots

24. Fig. Polymorphism comparison between the typical high-yield shikonin line AO-
30 (a) and low-yield shikonin line
Ai-4 (b). c Pearson correlation analysis between ET
concentration (Supplementary Fig. S7a) and shikonin
production (Supplementary Fig. S7b) of different hairy root
lines. Scatter diagram showing a significant positive linear
relationship between these two indexes (r = 0.9498; P0.001)

25. Catharantus roseus
‫بیان‬ ‫میزان‬ ‫بر‬ ‫اتیلن‬ ‫اثرات‬ ‫بررسی‬4‫ژن‬
(T16H,G16H,DAT,AVLBS)‫در‬‫گلدهی‬ ‫مرحله‬ ‫در‬ ‫پروانش‬ ‫گیاه‬
‫داد‬ ‫نشان‬.‫کشت‬ ‫در‬ ‫اتیلن‬ ‫غلظت‬ ‫افزایش‬ ‫که‬‫ویترو‬ ‫این‬‫تاثی‬‫قابل‬ ‫ر‬
‫بیان‬ ‫روی‬ ‫بر‬ ‫توجهی‬‫بیوسنتز‬ ‫مسیر‬ ‫در‬ ‫دخیل‬ ‫ژنهای‬TIA
‫آنها‬ ‫بیوسنتزی‬ ‫مسیر‬ ‫های‬ ‫آنزیم‬ ‫و‬ ‫آلکالوئید‬ ‫تولید‬ ‫روی‬ ‫بر‬‫د‬‫ارد‬.
‫افزایش‬ ‫باعث‬ ‫پروانش‬ ‫گیاه‬ ‫سلولی‬ ‫های‬ ‫لاین‬ ‫در‬ ‫اتیلن‬ ‫کاربرد‬
‫شد‬ ‫اجمالسین‬.(L.sayed et al 2004)
‫سرپنتین،ت‬ ،‫اجمالسین‬ ‫تولید‬ ‫افزایش‬ ‫باعث‬ ‫همچنین‬ ‫اتیلن‬‫ابرس‬
‫شد‬ ‫دولین‬ ‫وین‬ ‫و‬ ‫ونین،کاتارنتین‬.(yahya et al,2004)

26. • The yellow-fruited tomato 1 (yft1) mutant has
altered fruit carotenoid accumulation and reduced
ethylene production as a result of a genetic lesion
in ETHYLENE INSENSITIVE2(Tomato Research
Institute).

27. Repression of either MaMADS1 or MaMADS2, results in delayed ethylene synthesis and maturation,
though the fruit ripen normally when treated with ethylene. Banana fruits of control and MaMADS1 or
MaMADS2 repressed transgenic plants. Individual banana fruit are presented from each of the transgenic
antisense MaMADS2 and RNAi MaMADS2 and from control plants, and whole hands from RNAi
MaMADS1 and control plants. Fruits of the first and second hands were placed at 20oC and photographed
at 20 DAH and 26 DAH.

28. Molecular analysis of softening
and ethylene synthesis and
signaling pathways in a non-
softening apple cultivar,
‘Honeycrisp’ and a rapidly
softening cultivar,‘McIntosh’.

29. • Table: Transformation of ornamental plants modifying ethylene receptors

30. Expression of An Antisense Brassica oleracea GIGANTEA (BoGI)
Gene in Transgenic Broccoli Causes Delayed Flowering, Leaf Senescence, and
Post-Harvest Yellowing Retardation.

31. • Identifying and silencing tomato ripening genes with antisense genes
Figure : Knockout of the fruit-ripening-
specific
PG mRNA in transgenic plants using a half-
length antisense mRNA sequence under
control of the CaMV 35S promoter. This
result was first obtained at SB in the
autumn of 1987 and published in Nature by
Smith et al., 1988. Note that the antisense
mRNA bands (there are two of them, the
shorter one probably due to a fortuitous
internal polyA signal) accumulate before
ripening. When transcription of the PG
mRNA is switched
on, all three RNAs decline substantially in
amount. This is consistent with the
destruction of RNA-RNA hybrids formed
after PG mRNA transcripts start to
accumulate.

32. Gene
Introduced
Gene Source Product Function
anti-efe Lycopersicon
esculentum
antisense RNA of
1-amino-
cyclopropane -1-
carboxylate
oxidase (ACO)
gene (no
functional ACO
enzyme is
produced)
causes delayed
ripening by
suppressing the
production of
ethylene via
silencing of the
ACO gene that
encodes an
ethylene-
forming enzyme
Name: Huafan No 1
• Crop: Lycopersicon esculentum - Tomato
• Developer:
Huazhong Agricultural University (China)
• Method of Trait Introduction:
Microparticle bombardment of plant cells or tissue
• GM Trait :
Delayed ripening/senescence
• Commercial Trait:
(Singular) Modified Product Quality

33. Gene Introduced Gene Source Product Function
pg (sense or
antisense)
Lycopersicon
esculentum
no functional
polygalacturonase
enzyme is produced
(transcription of the
endogenous
enzyme is
suppressed by a
gene silencing
mechanism)
inhibits the
production of
polygalacturonase
enzyme responsible
for the breakdown of
pectin molecules in
the cell wall, and
thus causes delayed
softening of the fruit
• Trade Name: FLAVR SAVR™Developer:
Monsanto Company (including fully and partly owned companies)
• Method of Trait Introduction:
Agrobacterium tumefaciens-mediated plant transformation
• GM Trait s :
, Delayed fruit softening
Slow down ripening
not softe
Increased shelf life
antisense RNA

34. Gene
Introduced
Gene Source Product Function
pg (sense or
antisense)
Lycopersicon
esculentum
no functional
polygalacturonas
e enzyme is
produced
(transcription of
the endogenous
enzyme is
suppressed by a
gene silencing
mechanism)
inhibits the
production of
polygalacturonas
e enzyme
responsible for
the breakdown of
pectin molecules
in the cell wall,
and thus causes
delayed softening
of the fruit
Crop: Lycopersicon esculentum - Tomato
Developer:
Zeneca Plant Science and Petoseed Company
Method of Trait Introduction:
Agrobacterium tumefaciens-mediated plant transformation
GM Trait s :
Antibiotic resistance , Delayed fruit softening
Commercial Trait:
(Singular) Modified Product Quality
Summary of Basic Genetic Modification

35. Gene
Introduced
Gene Source Product Function
sam-k Escherichia coli
bacteriophage
T3
S-
adenosylmethion
ine
hydrolase enzym
e
causes delayed
ripening by
reducing the S-
adenosylmethioni
ne (SAM), a
substrate for
ethylene
production
Event Name: Melon A
Event Code : not available
Trade Name: not available
Crop: Cucumis melo - Melon, Cantaloupe
Developer:
Agritope Inc. (USA)
Method of Trait Introduction:
Agrobacterium tumefaciens-mediated plant transformation
GM Trait s :
Delayed ripening/senescence , Antibiotic resistance
Commercial Trait:
(Singular) Modified Product Quality

36. A reduction in total lycopene
accumulation was observed in EFE-
antisense fruit ripened
The rate of over-ripening and fruit
spoilage was also reduced in the
EFE-antisense fruitshowed that
the synthesis of ethylene during
fruit ripening was efficiently
inhibited by an EFE-antisense
transgene, and resistance to over-
ripening of fruit was reported.
Figure 1. Ethylene evolution from attached and detached wild-type and
EFE-antisense t m t o frud.
Wtld-type (A)o r EFE-antisensefruil( A)w ere left attached to the plant prior
to ethylene measurement or were detached at the mature-green stage and
npened in au (wild-type, m; EFE-antisense. 0). Ethylene was measured at
fruit npening stages mature-green (MG). breaker (Br) and 2. 4, 6 and 28
days post-breaker. Data are expressed as the mean of values obtained
from at least four fruit
Altered fruit ripening and leaf senescence
in tomatoes expressing an antisense
ethylene-forming enzyme transgene.

37. • an apple (Malus domestica) ethylene response factor, MdERF2,
negative affects ethylene biosynthesis and fruit ripening by
suppressing the transcription of MdACS1, a gene that is critical
for ripening-related ethylene biosynthesis. MdERF2 expression
was suppressed by ethylene during apple fruit ripening, and we
observed that MdERF2 bound to the promoter of MdACS1 and
directly suppressed its transcription. Moreover, MdERF2
suppressed the activity of the promoter of MdERF3, a
transcription factor that we found to bind to the MdACS1
promoter, thereby increasing MdACS1 transcription. We
determined that MdERF2 and MdERF3 proteins directly interact
and this interaction suppresses the binding of MdERF3 to the
MdACS1 promoter
•
Apple (Malus domestica) MdERF2 Negatively Affects Ethylene Biosynthesis
During Fruit Ripening by Suppressing MdACS1 Transcription

38. The use of antisense technology and overexpression of
metabolizing enzymes in controlling fruit ripening is
only the first step toward controlling fruit senescence.
Expression of antisense RNA using regulated promoters
may eliminate the use of exogenous ethylene for
reverting the mutant phenotype. However, the
development of gene transplacement technology by
homologous recombination should allow the creation of
nonleaky ripening mutants with long-term storage
potential. The prospect arises that inhibition of ethylene
production using reverse genetics
may be a general method for preventing senescence in a
variety of fruits and vegetables.

39. To be friends is quik work
But friendship is a slow ripening fruites
Thanks and Questions