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Bioscience Methods 2014, Vol.6, No.1, 1-13
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10
cells responds to water stress at the molecular level.
More importantly, plant genomes annotated have
revealed that a gene has diverse functions. Therefore,
to reveal a specific role of a gene or its product and its
interaction with other genes/proteins in a stress
response pathway, a prerequisite is to clarify and
specify the function(s) of the gene and any differences
from other genes with the same or similar functions.
Based on the consideration above and the genome
sequence database, starting with an EST from a
StOSM
gene isolated from the stress subtractive
library for water stress, the drought responses of
eleven
StOSM
genes were revealed. Eight
StOSM
s
were upregulated and three were downregulated in
response to water deficits. Using the RegSite Plant
Database and the online software TSSP, the binding
sites for
cis
-elements in the
StOSM
promoter were
analysed, revealing that the pattern of binding sites in
the
StOSM
promoters varies among the eleven
StOSM
genes. The resulting expression of the different
StOSM
genes varies during the response to water stress.
The drought tolerance capacity of a plant, similar to its
tolerance to other stresses, has been formed during the
long process of evolution. No water conditions except
extreme drought induce the expression of potential
drought responsive genes and the accumulation of
drought responsive molecules. Based on the potato
drought-lethal critical point (DLP) defined in this
study according to the phenotype under water deficit,
StOSM
mRNA accumulation reaches its peak at DLP
along a degressive gradient toward the DLP. This
observation has not been previously reported.
The qualitative and quantitative expression of the
StOSM
genes in this study is in agreement with the
data analysis of the potato genome sequence (Xu
et al.
2011). Osmotins, as response molecules, are clearly
involved in drought tolerance in the potato. However,
the extent to which the osmotins are associated with
drought tolerance in the potato, such as the specific
stages in the adult plant and during the restoration of
water after DLP, has yet to be determined. The
answers to these questions are indispensable to
establishing the dynamic network involved in drought
response.
In addition, osmotins, as drought responsive players,
surely interact or crosstalk with other molecules in the
drought response network. In this study, the
qualitative and quantitative results of StOSM in
response to drought provide a background in which
the interaction or crosstalk of StOSM with other
response molecules can be revealed to establish the
network.
Although the first
StOSM
(accession No. M29279)
was isolated almost 25 years ago, eleven-
StOSM
in
S.
tuberosum
identified in this study is the first one to
disclose OSM members in a plant genome. This is
necessary not only for understanding
StOSM
function
in the drought response network, but also for
engineering improvements in plant tolerance to
drought.
3. Materials and Methods
3.1 Plant materials
S. tuberosum
genotype ZHB is a tetriploid cultivar.
ZHB seedling can survival at the level of relative soil
water content as low as 10%. RH (RH89-039-16)
genotype is a diploid clone derived from a cross
between a
S. tuberosum
‘dihaploid’ (SUH2293) and a
diploid clone (BC1034). RH was selected as a diploid
model for potato genome sequencing. In this study
ZHB minitubers were sown in a pot containing peat as
the medium (Pindstruo Mosebrug, DK) and grown
under the following conditions: water content of 70%
±5% in the medium, light intensity of 100 µmol m
-2
s
-1
, temperature of 20-25°C and relative humidity of
60-80%, as it was done by
Anne J.K
et al.,
1996, Liu et al. 2012)).
On the 35th day after minituber germination
(when the
stored starch and other nutrition in the minituber is
completely exhausted as observed in our case), the
seedlings were kept under the same conditions, with
the exception that no water was provided. The water
content in the medium (WCM) was assayed every 12
hours until seedling death. Leaf sampling with three
replicates was performed along with a water deficit
gradient above the drought-lethal critical point (DLP)
ranging 70, 60, 50, 40, 30, 20 and 10 %. Ten-gram
leaf samples were collected, frozen in liquid nitrogen
and stored at –80°C for subsequent experiments.
3.2 Cloning of a
StOSM
with its EST sequence.
Total RNA was isolated from the leaf sample at
70%±5% and 20% ±2% WCM using
TRIZOL
Reagent
(15596-026,
Invitrogen
Corp., Carlsbad, CA, USA).