Abstract
This study aimed to investigate drought resistance of the LY1306
tobacco strain. Seedlings of tobacco strains LY1306, ZhongYan 100
(ZY100) and Hong Hua Da Jin Yuan (HHDJY) were treated with polyethylene
glycol (PEG)-6000 to induce osmotic stress. As validation,
water-deficit-induced drought treatments, including mild drought (MD;
watering 1.5 L/week) and severe drought (SD, without watering) were
carried out. Changes in cell morphology, leaf water potential,
antioxidant enzyme activity, as well as contents of malondialdehyde
(MDA) and proline were determined for each treatment. Transcriptome
sequencing was performed for the seedlings treated with 15% PEG-6000.
No obvious changes were observed in morphology of LY1306 and ZY100
under osmotic or drought stress; whereas, visible wilting was observed
in HHDJY. Superoxide dismutase and peroxidase activities of LY1036 and
ZY100 under osmotic stress were significantly higher than those of
HHDJY. Under SD, the MDA content of LY1306 was significantly lower and
the proline content of LY1306 was significantly higher than that of
HHDJY. Differential genes between LY1306, ZY100 and HHDJY were enriched
in functions about alpha-linolenic acid, and arginine and proline
metabolisms. LY1306 could increase its antioxidant enzyme activities
and proline accumulation in response to drought stress, probably by
regulating drought resistance-related pathways and genes.
Introduction
Drought is one of the most common environmental stresses and is
commonly defined as a period without significant rainfall^[32]1.
Drought severely constrains plant growth and productivity, which can
threaten agroforestry and lead to environmental deterioration^[33]2,
affecting both elongation and expansion growth at the initial phases of
plant establishment^[34]3,[35]4. Further, drought has adverse effects
on plant metabolic processes, including nutrient uptake, stomatal
movement and production of photosynthetic assimilates, which ultimately
results in crop losses^[36]1,[37]5,[38]6.
Tobacco (Nicotiana tabacum), an agriculturally important Solanaceae
crop, is one of the most studied plants as a biological model
system^[39]7. Importantly, it is a valuable economic crop and is the
most widely grown non-food crop worldwide^[40]8. Tobacco originates in
the tropics under conditions of good rainfall and requires ample water
for growth during development. Most tobacco crops entering the world
trade are produced in the temperate and tropical regions^[41]9.
According to a Food and Agriculture Organization report, in 2003, China
was one of the leading countries growing tobacco^[42]10. However,
currently, drought stress has become a main limiting factor for the
production of tobacco in China, particularly in northern China.
Therefore, breeding of drought-resistant tobacco varieties is an urgent
requirement.
LY1306 is a newly bred tobacco strain obtained through eight years of
hybridisation (2005–2012). Considerable field trials (2012–2015)
suggest that this strain has stable genetic traits, good yield and
quality and high stress and viral disease resistances. However, the
underlying physiological and molecular mechanisms have not been
investigated.
At the molecular level, most events involved in adaptation probably
result from alterations in gene expression^[43]11. Numerous studies
have applied the transcriptomic approach to investigate the drought
responses in plants^[44]12,[45]13, which have provided substantial
contributions to our understanding of the molecular mechanisms
underlying drought resistance. In the present study, we investigated
the drought resistance mechanisms of the LY1306 tobacco strain using
biochemical and transcriptomic approaches by comparing with another two
tobacco varieties, ZhongYan 100 (ZY100) and Hong Hua Da Jin Yuan
(HHDJY). ZY100 is a flue-cured tobacco variety developed by crossing
the female parent tobacco strain 9201 and the male parent variety NC82,
which presents good adaptability and drought resistanceis, and is
resistant to multiple diseases^[46]14. HHDJY is selected and bred from
the variant of Da Jin Yuan, which is superior in quality, but is
sensitive to drought^[47]15. Our data may provide important insight
into understanding the drought resistance mechanisms of the LY1306
tobacco strain.
Results
Effect of drought stress on morphological changes in LY1306
Under normal growth conditions, the growth of LY1306 was similar to
that of control strains (ZY100 and HHDJY). After being treated with 25%
PEG-6000 for 5 h, the leaves of HHDJY showed visible wilting, whereas
those of LY1306 and ZY100 remained normal (Fig. [48]1a). Moreover,
after treating seedlings with 15% PEG-6000 for 16 h, slight wilting was
observed in HHDJY, whereas no changes appeared in LY1306. After
continuous osmotic stress (15% PEG-6000) for 24 h, there was still no
obvious morphological change in LY1306. However, HHDJY exhibited severe
wilting (Fig. [49]1b). In addition to PEG-6000-induced osmotic stress,
we induced drought by withholding water supply. Similar to the findings
described above, LY1306 exhibited better drought resistance than HHDJY.
LY1306 seedlings in the mild drought (MD) group retained their leaf
morphology, whereas those in the severe drought (SD) group showed only
slight colour changes (Fig. [50]1c). For HHDJY, SD caused severe
wilting and the leaves turned deep green.
Figure 1.
[51]Figure 1
[52]Open in a new tab
The morphological responses of LY1306, ZhongYan 100 (ZY100) and Hong
Hua Da Jin Yuan (HHDJY) tobacco strains under drought stress. (a)
Seedlings treated with 25% PEG for 5 h; (b) Seedlings treated with 15%
PEG for 16 h or 24 h; (c) Seedlings subjected to mild drought (MD) and
severe drought (SD) treatments. (d) Changes of leaf water potential in
three vatieties after 15% PEG treatment for 0, 16 and 24 h. (e) Changes
of chlorophyll content in three vatieties after 15% PEG treatment for
0, 16 and 24 h. Results are expressed as the mean ± standard error from
three replication experiments. *P < 0.05, **P < 0.01 and ***P < 0.001
compared with the corresponding control groups.
In accordance with the findings above, water potential measurement
showed that water potential of HHDJY did not change obviously after
16 h of 15% PEG-6000 treatment but decreased significantly after 24 h
of treatment (P < 0.05), which suggested that HHDJY failed to respond
to osmotic stress quickly. On the contrary, LY1306 decreased leaf water
potential after 16 h of 15% PEG-6000 treatment (P < 0.01). Besides, the
water potential at 16 h was similar to that at 24 h (P > 0.05). For
ZY100, its water potential significantly increased after 16 and 24 h of
PEG-6000 treatment (P < 0.001), suggesting that ZY100 may have
different drought resistance mechanism with LY1306 (Fig. [53]1d).
Furthermore, the chlorophyll content of HHDJY was significantly higher
than that in LY1306 and ZY100 after 24 h of 15% PEG-6000 treatment
(P < 0.001) (Fig. [54]1e), which might explain the phenomenon that the
leaves of HHDJY turned deep green in SD group.
Effect of PEG-6000 on antioxidant enzyme activity of LY1306
The activities of antioxidant enzymes, superoxide dismutase (SOD),
peroxidase (POD) and catalase (CAT) after treatment with 15% PEG-6000
were investigated. LY1036 and ZY100 strains subjected to 15% PEG-6000
treatment exhibited significantly higher SOD activity (P < 0.05) than
the controls after 24 h of treatment (175.12% for LY1306 and 109.31%
for ZY100) (Fig. [55]2a). In addition, after 24 h of treatment, LY1036
and ZY100 exhibited significantly higher SOD activities (P < 0.01) than
HHDJY. These results suggest that SOD activity plays an important role
in drought resistance of LY1306.
Figure 2.
[56]Figure 2
[57]Open in a new tab
Changes in antioxidant enzyme activity of LY1306, ZhongYan 100 (ZY100)
and Hong Hua Da Jin Yuan (HHDJY) tobacco strains after treatment with
15% PEG-6000 for 16 h and 24 h. (a) superoxide dismutase (SOD); (b)
peroxide (POD) and (c) catalase (CAT). Results are expressed as the
mean ± standard error from three replication experiments. *P < 0.05,
**P < 0.01 and ***P < 0.001 compared with the corresponding control
groups.
Figure [58]2b presents changes in POD activities among the three
tobacco strains treated with 15% PEG-6000. The POD activity of LY1036
showed a 155.65% increase (P < 0.01), that of ZY100 showed a 6.35%
increase, whereas that of HHDJY showed a 31.07% decrease (P < 0.01)
compared with those of the corresponding control groups after 16 h of
treatment. Additionally, the POD activities of LY1036 and ZY100 were
significantly higher (P < 0.01) than that of HHDJY. After 24 h, POD
activities of all three tobacco strains significantly increased
(P < 0.001) compared with those of the corresponding controls groups
(321.03% for LY1306, 154.47% for ZY100 and 43.70% for HHDJY).
Furthermore, the POD activities were higher in LY1036 and ZY100 than in
HHDJY. Therefore, POD activity may be important for drought resistance
in LY1306.
There was no significant difference in CAT activities among the three
treatments groups in LY1306 (Fig. [59]2c). In contrast, in ZY100, the
CAT activity in the 15% PEG-6000 treatment group was significantly
lower (P < 0.05) than that in the control group. The CAT activity of
HHDJY increased at first and then decreased. No significant difference
was observed among the three tobacco strains after 24 h of 15% PEG-6000
treatment, suggesting that CAT activity does not influence drought
resistance of LY1306.
Effect of drought stress on MDA content of LY1306
MDA content is an important indicator of the degree of peroxidation in
plant cells. Variations in the MDA content were observed among the
three tobacco strains after treatment with 15% PEG-6000 and after
withholding water supply. After 16 h of 15% PEG-6000 treatment, the MDA
content of LY1306 significantly increased (P < 0.01) and that of HHDJY
slightly increased, whereas the MDA content of ZY100 decreased
(Fig. [60]3a). After 24 h, the MDA content of LY1306 decreased to the
same level as that exhibited by the corresponding MDA controls, whereas
the MDA content of ZY100 significantly decreased (P < 0.001) compared
with that of the corresponding MDA controls The MDA content of HHDJY
continuously increased throughout the experiment. In general, after
24 h, HHDJY had the highest (P < 0.001) MDA content among the three
strains. Under the water-deficit-induced drought stress, the MDA
content of LY1306 and ZY100 significantly increased (P < 0.05 or 0.01)
in MD group compared with control (Fig. [61]3b). Additionally, the MDA
content in SD group was significantly higher than that in control in
all of the three varities (P < 0.01). Moreover, the MDA content of
HHDJY was significantly higher than that of LY1306 and ZY100 in MD and
SD group (P < 0.01 or 0.001). These results suggest that MDA content is
a key indicator of drought resistance in LY1306.
Figure 3.
[62]Figure 3
[63]Open in a new tab
Changes in malondialdehyde (MDA) content of LY1306, ZhongYan 100
(ZY100) and Hong Hua Da Jin Yuan (HHDJY) tobacco strains after (a)
being treated with 15% PEG-6000 for 16 h or 24 h and (b) withholding
water supply. Results are expressed as the mean ± standard error from
three replication experiments. *P < 0.05, **P < 0.01 and ***P < 0.001
compared with the corresponding control groups.
Effect of drought stress on proline content of LY1306
Proline plays an important role in plant osmoregulation, and the
proline content changes under drought stress^[64]16. After 16 h of 15%
PEG-6000 treatment, compared with the controls, the proline content
increased by 280.78% in LY1306 (P < 0.001) and by 41.98% in HHDJY
(P < 0.001), whereas it decreased by 51.27% in ZY100 (P < 0.01). After
24 h of treatment, the proline content of LY1306 and HHDJY continuously
increased (P < 0.001) compared with those of the corresponding
controls. Further, the proline content of ZY100 increased after 24 h of
treatment (Fig. [65]4a). In addition, the proline content under drought
stress was higher (P < 0.05) in LY1306 and ZY100 than that in HHDJY.
When water supply was withheld, the proline content in the three tested
tobacco strains increased (Fig. [66]4b). Furthermore, under SD, the
proline contents of LY1306 and ZY100 were significantly higher
(P < 0.05) than that of HHDJY. These results suggest that proline plays
an important role in drought resistance of LY1306.
Figure 4.
[67]Figure 4
[68]Open in a new tab
Changes in proline content of LY1306, ZhongYan 100 (ZY100) and Hong Hua
Da Jin Yuan (HHDJY) tobacco strains after (a) being treated with 15%
PEG-6000 for 16 h and 24 h and (b) withholding water supply. Results
are expressed as the mean ± standard error from three replication
experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the
corresponding control groups.
Effect of drought stress on chloroplast ultrastructure in mesophyll cells
Under normal moisture conditions (CK), cell structure, chloroplasts
morphology and ultrastructure among the three strains were similar.
Under SD for 15 days, the number of chloroplasts decreased in all the
three strains; however, the number of chloroplasts in LY1306 was more
than that in HHDJY. Additionally, chloroplast morphology and thylakoid
structure of LY1306 cells were normal. In HHDJY, chloroplasts showed
shape change, and the lamellae of the thylakoids were disorganised
(Fig. [69]5).
Figure 5.
[70]Figure 5
[71]Open in a new tab
Chloroplast ultrastructure of mesophyll cells in LY1306, ZhongYan 100
(ZY100) and Hong Hua Da Jin Yuan (HHDJY) tobacco strains under severe
drought (SD) conditions. (a) cellular structure (2 μm); (b) chloroplast
ultrastructure (1 μm).
Sequence alignment
By mapping clean reads to the K326 tobacco reference genome, we
obtained total map rates of each of the three tobacco strains (LY1306,
89.81%; ZY100, 91.62% and HHDJY, 91.18%); after mapping the clean reads
to a K326 tobacco reference gene, total map rates of each of the three
tobacco strains were 95.39%, 96.20% and 95.96%, respectively.
Differentially expressed gene (DEG) analysis
In total, 3,066 DEGs including 1,449 up-regulated and 1,617
down-regulated genes, were identified between LY1306 and ZY100 strains.
Additionally, 3126 DEGs including 1,660 up-regulated and 1,466
down-regulated DEGs were identified between LY1306 and HHDJY
(Supplementary Table [72]1). Furthermore, 564 common up-regulated abd
434 common down-regulated DEGs were identified between LY1306 vs. ZY100
and LY1306 vs. HHDJY.
Functional enrichment analyses
Gene ontology (GO) enrichment analysis showed that the up-regulated
DEGs between LY1306 and ZY100 were mainly enriched with genes
regulating biological processes associated with cinnamic acid,
terpenoid and oxylipin biosyntheses. DEGs between LY1306 and HHDJY were
mainly enriched with genes regulating biological processes, including
oxylipin, terpenoid and jasmonic acid biosyntheses, wounding response
and lipid oxidation. Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway enrichment analysis revealed that the up-regulated DEGs between
LY1306 and ZY100 were mainly enriched with genes regulating
alpha-linolenic acid, linoleic acid, arginine and proline metabolisms.
Additionally, these pathways were also enriched by DEGs between LY1306
and HHDJY (Fig. [73]6a and b). Furthermore, the common up-regulated
DEGs were significantly enriched in GO terms associated with lipid and
oxylipin biosyntheses and metabolisms, and pathways regulating
alpha-linolenic acid, linoleic acid [such as mRNA_139072, mRNA_139073,
and mRNA_139074 (all the three genes encoded linoleate 13S-lipoxygenase
2-1, chloroplastic-like, partial)], and arginine and proline
metabolisms [such as mRNA_107515 (polyamine oxidase 1 isoform ×2),
mRNA_109766 (putative amidase C869.01), and mRNA_142147
(S-adenosylmethionine decarboxylase proenzyme-like)] (Fig. [74]6c).
Figure 6.
[75]Figure 6
[76]Open in a new tab
(a) Biological processes and KEGG pathways enriched by the up-regulated
differentially expressed genes between LY1306 and ZhongYan 100 (ZY100)
tobacco strains after being treated with 15% PEG-6000 for 16 h. (b)
Biological processes and KEGG pathways enriched by the up-regulated
differentially expressed genes between LY1306 and Hong Hua Da Jin Yuan
(HHDJY) tobacco varieties after being treated with 15% PEG-6000 for
16 h. (c) Biological processes and KEGG pathways enriched by the common
up-regulated differentially expressed genes of LY1306 vs. ZhongYan 100
(ZY100) and LY1306 vs. Hong Hua Da Jin Yuan (HHDJY). (d,e and f)
Biological processes and KEGG pathways enriched by the down-regulated
differentially expressed genes.
In addition to up-regulated DEGs, the down-regulated DEGs of LY1306 vs.
ZY100 were enriched in GO terms related to metal ion transport, and
ammonium transport, as well as pathways of Glycolysis/Gluconeogenesis,
and Starch and sucrose metabolism. The down-regulated DEGs of LY1306
vs. HHDJY were enriched in GO terms about floral organ abscission, and
carpel development, and pathways of Protein processing in endoplasmic
reticulum and Ribosome biogenesis in eukaryotes. Moreover, the common
down-regulated DEGs of LY1306 vs. ZY100 and LY1306 vs. HHDJY were
significantly involved in GO functions related to DNA integration, and
pathway of Photosynthesis-antenna proteins.
Our biochemical experiments showed that proline plays an important role
in drought resistance of LY1306, therefore, we displayed the pathway
map of arginine and proline metabolisms (Fig. [77]7). In the KEGG map,
1.5.3.14 indicates mRNA_107515 (polyamine oxidase 1 isoform ×2);
3.5.1.4 indicates mRNA_109766 (putative amidase C869.01); 4.1.1.50
represents mRNA_142147 (S-adenosylmethionine decarboxylase
proenzyme-like).
Figure 7.
[78]Figure 7
[79]Open in a new tab
Regulatory network of arginine and proline metabolisms. Green boxes and
yellow box represent the enriched DEGs (green includes up-regulated
gene, yellow includes both up-regulated and down-regulated genes). The
pathway map without coloring is the original version that is manually
drawn by in-house software named KegSketch. White boxes are hyperlinked
to KO, ENZYME, and REACTION entries in metabolic pathways, and to KO
and GENES entries in non-metabolic pathways. Purple boxes are
hyperlinked to KO entries that are selected from the original version.
Discussion
The present study investigated the drought resistance ability of a
newly bred tobacco strain, LY1306, and further explored the potential
mechanisms underlying the drought stress resistance using biochemical
and transcriptomic approaches. Our results revealed that LY1306
exhibited better drought resistance than HHDJY. Our transcriptome
sequencing analyses revealed thousands of DEGs between LY1306 and two
control tobacco strains (ZY100 and HHDJY). These DEGs were
significantly enriched in chemical compounds involved in biosynthetic
and metabolic processes associated with GO terms and KEGG pathways.
Many of these chemical compounds may be responsible for the drought
resistance of LY1306.
Drought is a major environmental determinant of plant growth and
productivity. Exposure to drought stress induces the generation of
reactive oxygen species, which have a negative oxidative effect on
cellular structures and metabolism^[80]17. Plants have developed some
strategies to cope with this challenge. The antioxidant defence system
is one of the drought stress defence mechanisms^[81]18. SOD converts
O[2] ^− into H[2]O[2], POD reduces H[2]O[2] to H[2]O based on the
various substrates available as electron donors and CAT dismutates
H[2]O[2] into H[2]O and O[2]. Many studies have suggested that the
activities of antioxidant enzymes are correlated with a plant’s drought
resistance^[82]19,[83]20. The activities of antioxidant enzymes are
higher in stress-resistant species than in stress-sensitive
species^[84]21. Our data revealed that SOD and POD activities of LY1306
and ZY100 were significantly higher than those of HHDJY under stress
induced with 15% PEG-6000. Interestingly, there was no significant
change in CAT activity of LY1306 under the various treatments.
MDA is a by-product of lipid peroxidation, which serves as suitable
marker for membrane lipid peroxidation^[85]22, and the MDA content
usually increases under stress damage^[86]23. In general,
stress-sensitive tobacco strains have a higher MDA content and
electrolyte leakage in response to environmental stress than
stress-resistant strains^[87]24. In the present study, the MDA content
gradually increased with increased drought stress in all the three
strains. Interestingly, we also found that the MDA content of HHDJY was
significantly higher than those of LY1306 and ZY100 under both MD and
SD.
Proline can accumulate to high concentrations without damaging cellular
macromolecules. Therefore, it acts as a compatible osmolyte.
Importantly, proline provides protection against membrane damage and
protein denaturation during severe drought stress^[88]25.
Stress-mediated changes in free proline levels have been studied in
various plant species, and several possible roles conferring stress
resistance have been proposed^[89]26. Proline accumulation is a
physiological response of plants subjected to drought stress^[90]27.
Our results regarding proline accumulation were in agreement with the
results of studies cited above. In the present study, proline
accumulation in response to drought was observed in all the three
strains. In particular, proline contents of LY1306 and ZY100 were
significantly higher than that of HHDJY. Interestingly, our pathway
enrichment analysis revealed that the DEGs between LY1306 and ZY100 and
between LY1306 and HHDJY, such as mRNA_107515, mRNA_109766, and
mRNA_142147, were up-regulated in LY1306 and significantly enriched in
arginine and proline metabolisms. Therefore, proline accumulation in
LY1306 under drought stress may be associated with mRNA_107515,
mRNA_109766, and mRNA_142147 as well as arginine and proline
metabolisms.
Plant resistance to drought involves not only morphological and
biochemical adaptations but also responses at the genetic level^[91]28.
In the present study, numerous DEGs were identified between LY1306 and
ZY100, and between LY1306 and HHDJY. Some of these DEGs, such as
mRNA_139072, mRNA_139073, and mRNA_139074 (all the three genes encoded
linoleate 13S-lipoxygenase 2-1, chloroplastic-like, partial), are
significantly involved in alpha-linolenic acid and linoleic acid
metabolisms; these acids are important polyunsaturated fatty acids
(PUFAs) associated with cell membrane lipids. Lipids are important
membrane components and are severely affected by drought stress. Lipid
composition changes maintain membrane integrity under water stress
conditions^[92]29. In the biochemical experiments conducted in our
study, the MDA content was hypothesised to be a key indicator of
drought resistance of LY1306. MDA appears to be the most mutagenic
product of lipid peroxidation^[93]30. In general, lipid peroxidation is
described as a process under which oxidants attack lipids containing
carbon–carbon double bond(s), particularly PUFAs^[94]31. Therefore,
alpha-linolenic acid and linoleic acid metabolisms as well as their
enriched genes (mRNA_139072, mRNA_139073 and mRNA_139074) may be
associated with the MDA content in all the three tobacco strains.
Furthermore, our GO enrichment analysis found that these up-regulated
DEGs were mainly enriched in genes that regulate biochemical processes
associated with cinnamic acid, and jasmonic acid biosyntheses. Cinnamic
acid identified from the root exudates of cucumber^[95]32 could inhibit
plant growth by affecting ion uptake and hydraulic conductivity.
Cinnamic acid has been reported to reduce lipid peroxidation and
increase activities of antioxidant enzymes, such as SOD and POD, in
drought-stressed cucumber leaves^[96]33,[97]34. Jasmonic acid,
belonging to a class of derived polyunsaturated fatty acid
phytohormones, occurs ubiquitously in plants and induces a wide range
of plant stress responses. In particular, jasmonic acid is effective in
protecting plants from drought-induced oxidative damage^[98]35. A
recent study has reported that jasmonic acid can modulate the
antioxidant response mechanism of higher plants by tightly regulating
biomembrane peroxidation^[99]36. Therefore, we speculated that the
higher antioxidant enzymes activities in LY1306 in response to drought
stress are associated with cinnamic acid and jasmonic acid
biosyntheses.
Although our study investigated drought resistance mechanisms of LY1306
at the transcriptomic level and identified some DEGs and pathways that
may play roles in drought resistance of LY1306, the relative expression
of DEGs or the possible involvement of biochemical pathways in
regulating the expression of DEGs was not further investigated.
Therefore, our future study will focus on relevant research regarding
these factors.
In conclusion, our study suggests that the LY1306 strain could
significantly enhance the activities of antioxidant enzymes, increase
the accumulation of proline and decrease the MDA content in plant cells
as an adaptive response to drought stress. These drought resistance
reactions may be associated with biochemical pathways of arginine,
proline, alpha-linolenic acid and linoleic acid metabolisms, and
cinnamic acid and jasmonic acid biosyntheses.
Methods
Plant materials and growth conditions
The LY1306 tobacco strain (provided by Luoyang tobacco company, Henan,
China) and two control tobacco strains, ZhongYan 100 (ZY100) and Hong
Hua Da Jin Yuan (HHDJY) (both provided by Henan Agricultural
University, Henan, China) were used as biomodels in the present study.
Tobacco seedlings were grown in a floating system for 46 days in a
culture room (27 °C, 70% humidity and 16 h light).
Drought treatment
For polyethylene glycol (PEG)-6000-induced osmotic stress, tobacco
seedlings were transplanted into the perlite-based Murashige and Skoog
(MS) medium (without sugar and agar) when they grew four leaves. The
culture solution was changed every 3 days. After 23 days
post-transplantation, either 25% or 15% PEG-6000 was added to the MS
medium to induce osmotic stress for 5 h, 16 h or 24 h.
For water-deficit-induced drought stress, tobacco seedlings were
transplanted into culture pots and grown in nutrient-amended soil.
During the 35-day post-transplantation growth period, all seedlings
were watered once a week (3 L at each time). After 40 days
post-transplantation, the tobacco seedlings were randomly divided into
CK, MD and SD groups. The control group seedlings were watered once
every 5 days with 3 L of water; the MD group seedlings were watered
once every 5 days with 1.5 L of water; and the SD group seedlings were
not watered. All treatments lasted for 15 days. Then, watering was
discontinued for all seedlings until visible wilting symptoms appeared
in the drought treatment groups, which occurred within 20 days after
watering ceased.
Measurements
The changes in plant morphology, leaf water potential, chlorophyll
content, antioxidant enzyme activity, MDA and proline contents and
chloroplast ultrastructure in mesophyll cells of leaves in response to
osmotic or drought stress were observed. For each measurement, three
seedlings were randomly selected from each tobacco strain; leaves were
used for all measurements. All the experiments were repeated thrice.The
leaf water potential was detected with a pressure chamber (PMS
Instruments, Corvallis, OR).
Chlorophyll contents were determined with alcohol. Briefly, the fresh
leaves (0.2 g) were homogenized with 2-3 ml of 95% alcohol. Then 10 ml
alcohol was added into the homogenate and ground until the tissue
blanched. After 3 to 5 minutes’ standing, the extract liquid was
filtered into brown volumetric flask (25 ml). The funnel, filter paper
and mortar were washed with alcohol and liquid was added into the
volumetric flask. Finally, alcohol was added to make 25 ml. The
absorbance was measured at 665 nm, 649 nm and 470 nm, and chlorophyll
concentrations were calculated as follows:
[MATH: Chlorophylla:Ca(mg/l)=13.95×D665−
6.88×D649
:MATH]
[MATH: Chlorophyllb:Cb(mg/l)=24.96×D649−7.32×D665
:MATH]
[MATH: Carotenoid=(1000×D470−
2.05×Ca−114.8∗Cb)/245 :MATH]
Chlorophyll content was calculated as:
Chlorophyll content (mg/g) = chlorophyll concentration (mg/l) × volume
of extract liquid/Dry weight of the sample (g)
SOD, POD and CAT activities were determined using SOD, POD and CAT
microdetermination kits (Suzhou Comin Biotechnology Co., Ltd, Jiangsu,
China), respectively. MDA and proline contents were also estimated
using microdetermination kits (Suzhou Comin Biotechnology Co., Ltd).
The chloroplast ultrastructure of mesophyll cells was observed via
transmission electron microscope detection^[100]37. Briefly, after
sectioning, leaf samples (10 mm^2 in size) were fixed in phosphate
buffer (pH 7.2) containing 5% glutaraldehyde, followed with washing
with 0.1 molL^−1 phosphate buffer (pH 7.0) thrice. The washed samples
were then fixed for 2 h in 1% osmic acid and prepared and washed three
more times with the same phosphate buffer (pH 7.0). The washed samples
were dehydrated with 30%, 50%, 70%, 90% and 100% ethanol and then
placed into an embedding medium. The samples were then sectioned with a
Leica EM UC7 ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany)
and treated with uranyl acetate, followed by lead citrate. The samples
were examined and photographed using an electron microscope (H-7650;
Hitachi Ltd, Tokyo, Japan).
Transcriptome sequencing
Transcriptome sequencing was performed on mRNAs obtained from the
leaves of LY1306, ZY100 and HHDJY strains treated with 15% PEG-6000 for
16 h (Shanghai OE Biotech. Co., Ltd, Shanghai, China). Sequencing was
performed on three leaves from each strain. Total RNAs were extracted
from leaf tissue using mirVana™ miRNA ISOlation Kit (Ambion-1561), and
DNA was digested with DNase. mRNAs were isolated from 50 μL total RNA
using magnetic beads with an Oligo-dT tag. mRNAs were cleaved into
short fragments, which were then used as templates to synthesise
first-strand cDNA with random hexamer primers using the First Strand
Synthesis Act D Mix and SuperScript II Reverse Transcriptase (Cat. No.
18064014; Invitrogen, CA, USA). Double-stranded cDNA was synthesised
with the first-strand reaction using TruSeq Stranded mRNA LTSample
Preparation Kit (Cat. No. RS-122-2101; Illumina, CA, USA). After
purification of the double-stranded cDNA with the Agencourt AMPure XP
system (Cat. No. A63881; Beckman Coulter, Brea, CA, USA), end repair,
dA tailing and adaptor ligation were performed. Then, the end products
were size-selected and enriched using PCR to create a cDNA library for
transcriptome sequencing. Agilent 2100 Bioanaylzer (Agilent
Technologies, CA, USA) was used to quantify and control the quality of
the sample library. The paired-end libraries were then sequenced on the
Illumina HiSeq^TM 2500 platform.
All data are available at the National Center for Biotechnology
Information (NCBI) Sequence Read Archive database under the accession
number SRP100649.
Sequence alignment and gene expression analysis
The raw reads obtained from Illumina HiSeq^TM 2500 sequencing were
processed by removing the low-quality reads, and reads with N. The
obtained clean reads were aligned with the K326 tobacco reference
genome
(ftp://ftp.solgenomics.net/genomes/Nicotiana_tabacum/assembly/Ntab-K326
_AWOJ-SS.fa.gz) and a reference gene
(ftp://ftp.solgenomics.net/genomes/Nicotiana_tabacum/annotation/Ntab-K3
26_AWOJ-SS_K326.mrna.annot.fna) using TopHat software^[101]38 and
Bowtie2^[102]39, respectively.
The gene expression level is directly reflected in the abundance of its
transcript. Abundances were reported in expected fragments per kilobase
of transcript per million fragments mapped (FPKM)^[103]40. FPKM is
calculated based on the mapped transcript fragments, transcript length,
and sequencing depth, which is the most commonly used method for
estimating gene expression. In this study, we used reference genes to
measure the abundance of each transcript according to sequence
similarity alignment by using the software packages of Bowtie2^[104]39
([105]http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml) and
eXpress
([106]http://www.rna-seqblog.com/express-a-tool-for-quantification-of-r
na-seq-data/).
DEGs and functional enrichment analyses
Based on the obtained gene expression levels, DEGs between LY1306 and
ZY100 tobacco strains and between LY1306 and HHDJY tobacco strains were
screened using the DESeq package^[107]41.
([108]http://bioconductor.org/packages/release/bioc/html/DESeq.html)
with an adjusted probability criterion of P ≤ 0.05.
The identified DEGs were subjected to the GO function and KEGG pathway
enrichment analyses. The significance of enriched DEGs as per the GO
terms or KEGG pathway maps was calculated according to their
hypergeometric distributions. Any term or pathway that contained less
than three genes was removed.
Additionally, due to the important role of proline in drought
resistance, we downloaded the pathway map of arginine and proline
metabolisms from KEGG PATHWAY database. In the KEGG map, green boxes
and yellow box represent the enriched DEGs (green includes up-regulated
gene, yellow includes both up-regulated and down-regulated genes). The
pathway map without coloring is the original version that is manually
drawn by in-house software named KegSketch. White boxes are hyperlinked
to KO, ENZYME, and REACTION entries in metabolic pathways, and to KO
and GENES entries in non-metabolic pathways. Purple boxes are
hyperlinked to KO entries that are selected from the original version.
Statistical analysis
Our results of biochemical experiments are presented as mean ± standard
error. Statistical analyses were performed using SPSS 17.0. Differences
were compared using analysis of variance (ANOVA) followed by least
significant difference (LSD). P < 0.05 was considered as significant.
Electronic supplementary material
[109]supplementary table 1^ (1.5MB, xlsx)
Acknowledgements