Ethylene Perception and the Fruit bowl

portertoaststicksBiotechnology

Oct 23, 2013 (3 years and 7 months ago)

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1

Ethylene Perception and the
Fruit

bowl

Rebecca Gunsto
n.
In Biblical times, Amos was the
‘piercer of fig trees’ (Amos 7:14)

[1]
,
which was an extremely responsible
job. This required him to make an
incision in the fruit a few days before
harvest to encourage ripening. King
David even appointed an overseer
for
this tedious, yet vitally important task.
Now more than 2,000 years later, we
are beginning to understand the
science behind it.

Ethylene, a gaseous phytohormone, is
a major signalling molecule in plants.
It controls pathways causing seed
germination,
abscission, ripening,
senescence, and wound healing.
Control of this pathway is a billion
-
dollar business.

Previous research has identified four
separate genes responsible for
ethylene perception. This included the
etr1

gene which, when mutated,
causes an
80% reduction in ethylene
perception and response. In 1997,
Wilkinson
et al

[2]

reported that the
mutant gene
etr1
-
1

protein, initially
discovered by Schaller and Bleeker

[3]
,
confers ethylene
-
insensitivity in
heterologous plants. This was observed
phenotypically by the lack of the so
-
called ‘t
riple response’. Seedlings
grown in dark conditions and in the
presence of ethylene normally have
short, radially expanded roots and
hypocotyls, and exaggerated curvature
on the apical hook. In
etr1
-
1

mutants,
this response is decreased.

Wilkinson and co
-
workers

[2]

engineered two forms of the
Arabidopsis thaliana

etr1
-
1

sequence,
and transformed them into Kanomycin
-
resistant tomato and petunia plants,
mediated by
Agrobacterium
. The
vectors used allowed the exchange of
the N
-
terminus of the tomato ethylene
receptor f
or the mutant
Arabidopsis

protein. A similar process occurred in
petunia. They grew the plants, plus
wild type controls, in dark conditions,
on ACC
-
containing agar plates. The
conversion of ACC to ethylene by ACC
Oxidase results in the triple response
phen
otype, but the transformed plants
failed to demonstrate this. The
presence of the transgene was
demonstrated by PCR analysis.
T
able
1

shows the resultant phenotypes.
T
able
1
. Two of the most biotechnologically
-
important phenotypes, and the differences
between the Wild Type and the etr1
-
1 mutant, as observed in experiments by Wilkinson
et al (1997).

Phenotype

Tomato
-
specific characteristic

Petunia
-
specific characteristic

Wild Type

Mutant

Wild Type

Mutant

Flower
Senescence

Dry and
detached after
3
-
5 days

Remained
expanded and
attached for
more than 10
days. Removed
when physically
separated by
the ovary.

Collapsed by day
3

Turgid and
intact for
at
least 8 days

Fruit ripening

Turned deep
red, soft and
started to rot

Remained
golden yellow
after 3 months

N/A

N/A




2

The conclusions of this research have
several applications for biotechnology.
The ethylene pathway is highly
conserved between species and the
success of Wilkinson
et al

[2]

transgenic experiments indicate that
genetic manipulation of the ethylene
signalling pathway may be possible in
many species, including mo
nocots. It
may not be effective at eliminating
responses in all plants, but paves the
way for the production of crops with a
longer shelf life and of better quality,
therefore more desirable for
consumers, with less wastage for
farmers. Its applications in

the
floriculture industry are also extremely
beneficial. Currently, the treatment of
flowers with sodium thiosulfate
extends their lifetime because it
interferes with the ethylene signal
pathway. However, it is an
environmentally unfriendly method
and gen
etic manipulation will increase
flowering time, reducing the cost of
cut flower storage, making it more
beneficial for consumers.

Advances in this field of research have
been made due to the increased use of
genetic manipulation and the use of
epistatic an
alysis to deduce the
pathway, as shown in
Figure
1
.

Many questions remain unanswered,
the majority relating to specific
component
-
component interactions, or
particula
r component functions,
including EIN5, EIN6, EIN7, and the EIL
-
family.

Genetically modified crops are
becoming increasingly common
-
place,
despite the continuing debate over
their safety. Monsanto recently
revealed plans to increase the
worldwide area farmi
ng transgenic
crops by 44%. However, it has been
observed that the
etr1
-
1

gene is
expressed in a cell
-
autonomous
manner
[4]
. Nonuniform expression of
the transgenic gene will initiate
uneven ripening, causing a “distinct
ripening sector within an otherwise
non
-
ripening fruit.”
[2]
. This must be
borne in mind when considering the

breadth of this technique.

Figure
1

taken from

Chang and Shockey
(1999)

[5]
, demonstrating the current
knowledge of the ethylene perception
pathway. EIN2 is membrane bound, and
thus may associate with the nuclear
membrane, although this remains
elusive.


The pro
teins in the ethylene pathway
exhibit a high level of redundancy. If it
is shown that they are expressed in a
tissue
-
specific manner then mutation
of one gene may knockout ethylene
perception in one tissue, rather than
the whole plant. This may be
benefici
al for the prevention of
ethylene
-
controlled post
-
harvest
disorders in many crops. It may also be
extended to floriculture, crop viability
and storage length, and a practicable
way of controlling climacteric fruit
ripening. Current methods for
controlling
ethylene activity, such as
controlled greenhouse environment,
could be successfully combined with
gene manipulation to create a
reduction in ethylene sensitivity,
providing greater control over
ethylene
-
managed processes. This
would be more effective than
producing completely insensitive
mutants that could not be controlled.
Nevertheless, ethylene
-
producing
tomatoes, for example, must be
physically segregated from crops like
lettuces and cucumbers because their
ethylene mutation can be reversed in
the prese
nce of ethylene.



3

Another cautionary note must be
heeded, as deleterious alterations in
disease susceptibility to usually non
-
pathogenic soil fungi, and secondary
responses to infection, have been
observed in experiments conducted by
Chang and Shockey

[5]
. Other changes
to growth, fertility and response to
environmental stresses must also be
carefully analysed before the use
of
s
uch GM crops can be considered.

After more than two millennia, it
appears that Amos’ highly respected
job
may
no longer be necessary.

References

1.

Theologis, A.,
One rotten apple
spoils the whole bushel: the
role of ethylene in
fruit
ripening.

Cell, 1992.
70
: p.
181
-
184.

2.

Wilkinson, J.Q., et al.,
A
dominant mutant receptor
from Arabidopsis confers
ethylene insensitivity in
heterologous plants.

Nature
Biotechnology, 1997.
15
: p.
444
-
447.

3.

Schaller, G.E. and A.B.
Bleecker,
Ethy
lene
-
binding
sites generated in yeast
expressing the Arabidopsis
ETR1 gene.

Science, 1995.
270
:
p. 1809
-
1811.

4.

Tiemann, D.M. and H.J. Klee,
Differential expression of two
novel members of the tomato
ethylene
-
receptor family.

Plant
Physiology, 1999.
120
:
p. 165
-
172.

5.

Chang, C. and J.A. Shockey,
The ethylene
-
response
pathway: signal perception to
gene regulation.

Current
Opinion in Plant Biology, 1999.
2
: p. 352
-
358.