Molecular Plant Breeding2016, Vol.7, No.18, 1
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seedling tolerance to low temperature stress.
L. apetalum
shows special responsible characteristics to temperature during the germination. In laboratory,
L.
apetalum
seeds cannot germinate at low temperature (0°C-4°C), but germinate at 10°C to 25°C (Meng et al.,
2008). However, in the field,
L. apetalum
seeds could germinate at 2°C-3°C, the average temperature of early
spring in Northern Xinjiang, it shows a good tolerance to low temperature stress. Why
L. apetalum
seeds can
germinate at low temperature in early spring but cannot germinate at low temperature in the laboratory? The study
of Zhao Huixin (2010) pointed out that, although
L. apetalum
seeds cannot germinate at 4°C in the laboratory, it
can tolerate low temperature before and after a critical physiological stage, the stage before seeds just sprout. If
the temperature is low in early spring, there will be a stagnancy stage in early germination of
L. apetalum
seeds
after it imbibes moisture, but this stagnancy will be broken if there is a certain time of rise in the temperature. It is
consistent with the experimental results that appropriate stratification at low temperature in laboratory can
significantly improve germination energy of
L. apetalum
seeds, and the result that
L. apetalum
seeds can
germinate at 4 °C only after be treated for 1 h at 25°C after 10 d’s stratification. In addition,
L. apetalum
seeds
treated by cold induction in various periods over low temperature stagnancy has stronger tolerance to -5°C, -10°C
stress (Meng et al., 2008). How can
L. apetalum
seeds tolerate low temperature to germinate? To analyze the
physiological mechanism of this, Yang Na (2015) have studied and reported that, in the process of
L. apetalum
seeds germination, low temperature had a great effect on the activity of superoxide dismutase (SOD) and catalase
(CAT). Zhou Qian (2016) sequenced the transcriptome of
L. apetalum
seeds before and after germination
stagnancy at low temperature, the result showed that 159 genes had significant differences in expression, and 54
genes expression was up-regulated significantly. While the molecular mechanism of
L. apetalum
seeds tolerate
low temperature to germinate has not been reported at protein level. This study screened proteins responding to
temperature related to germination stagnancy and removement of germination stagnancy, then identified important
differential proteins, finally, discussed these proteins’ function, gene expression patterns in the process of
removing germination stagnancy, and analyzed the correlation between their expression quantity and germination
stagnancy, to lay the basis for clarifying the mechanism of
L. apetalum
seeds germination stagnancy at low
temperature.
1 Results
1.1Changes in total protein during germination of
L. apetalum
seeds
Total protein was extracted respectively from three groups of
L. apetalum
seeds with TCA-Acetone precipitation
methods, and protein content was measured respectively. The results showed, the total protein content in the first
group of seeds (The seeds without stratification) has reached to 3.46%, was the highest in three groups, which
may contain a large amount of storage protein. The total protein content in the second group of seeds (Theseeds
stayed at stage of germination stagnancy after stratification for 10 d at low temperature) has decreased to 2.91%
significantly. May be in the initial germination period, a large number of storage protein was decomposed to
provide energy for germination or changed into other non-protein substances for the need of germination. The
total protein content in the third group of seeds (The seeds removed stagnancy after treated for 50 min at 25°C)
has reached to 3.19%, was significantly increased than the second group. It showed that there were some new
proteins synthesized in the process of removing germination stagnancy, which may be closely related to the
germination of
L. apetalum
seeds at low temperature.
1.2 Screening for temperature-responding proteins during germination of
L. apetalum
seeds
There were about 600 protein spots were separated by two-dimensional gel electrophoresis (2-DE) in every
groups of seeds. We found 37 distinct protein spots (Figure 1) in the 2-DE map of the third group different from
the other two groups, of which 14 of them were up regulated and 23 down regulated. 6 up-regulated proteins (24,
27, 28, 29, 31, 32) were identified by LC-MS/MS, and the result was analyzed in Swissprot and NCBI database
with MASCOT. These 6 proteins is molecule chaperon protein Cpn60, heat shoct proteins Hsp17.6I and Hsp70B,
cell division control protein 48 homolog E (Cdc48E), peroxidase 12 (Per12) and peroxidase 28 (Per28) of
oxidoreductase family
Ⅲ
respectively. As a molecular chaperone, Cpn60 involved in protein folding, and help
related proteins play a corresponding role. When the seed subjected to heat shock, osmotic shock or salt stress in
