Abstract
Drought stress is a serious problem worldwide that reduces crop
productivity. The laser has been shown to play a positive physiological
role in enhancing plant seedlings tolerance to various abiotic
stresses. However, little information is available about the molecular
mechanism of He-Ne laser irradiation induced physiological changes for
wheat adapting to drought conditions. Here, we performed a large-scale
transcriptome sequencing to determine the molecular roles of He-Ne
laser pretreated wheat seedlings under drought stress. There were
98.822 transcripts identified, and, among them, 820 transcripts were
found to be differentially expressed in He-Ne laser pretreated wheat
seedlings under drought stress compared with drought stress alone.
Furthermore, most representative transcripts related to photosynthesis,
nutrient uptake and transport, homeostasis control of reactive oxygen
species and transcriptional regulation were expressed predominantly in
He-Ne laser pretreated wheat seedlings. Thus, the up-regulated
physiological processes of photosynthesis, antioxidation and osmotic
accumulation because of the modified expressions of the related genes
could contribute to the enhanced drought tolerance induced by He-Ne
laser pretreatment. These findings will expand our understanding of the
complex molecular events associated with drought tolerance conferred by
laser irradiation in wheat and provide abundant genetic resources for
future studies on plant adaptability to environmental stresses.
Introduction
Wheat is one of the most important staple crops in the world by virtue
of its key contribution to food security. Drought stress severely
limits crop production and reduces the yield and quality of
wheat^[32]1. Importantly, drought stress is predicted to become more
frequent and severe due to the future climate change, and thus poses a
serious challenge to agricultural production worldwide^[33]1, [34]2.
Therefore, understanding the molecular mechanisms responsible for plant
drought stress tolerance is essential for improving this beneficial
trait in crops.
With the development of laser technology, laser has been widely used in
the field of biology. Low dose of laser irradiation can promote plant
growth and physiological metabolism^[35]3–[36]5 and also protect plant
seedlings against enhanced UV-B radiation^[37]6, chilling stress^[38]7,
osmotic stress^[39]8, drought stress^[40]9, and cadmium stress^[41]10.
Laser irradiation enhanced plants tolerance mainly through improving
seeds germination rates, plant height, root length, seedlings biomass,
photosynthesis, better maintenance of reactive oxygen species (ROS)
homeostasis and membrane stability at the physiological level in
crops^[42]11, [43]12. Gao et al. have illustrated that He-Ne alleviated
the adverse impacts of salt stress on plant growth by enhancing
relative water content, and meanwhile, increasing chlorophyll
concentrations and the activity of photosystem II resulting in
improvement of plant photosynthetic activity^[44]11. Our previous
investigations showed that He-Ne and CO[2] laser pretreatment could
enhance drought tolerance of wheat seedlings by increasing the
concentration of ascorbic acid (AsA) and glutathione (GSH) and the
activities of enzymes involved in reactive oxygen species (ROS)
scavenging including superoxide dismutase (SOD), peroxidase (POD),
catalase (CAT), ascorbate peroxidase (APX)^[45]8, [46]9. Although much
effort has focused on elucidating the mechanism of action of laser
irradiation at morphological and physiological levels, such as seed
germination, photosynthetic rate, chlorophyll concentration,
antioxidant compounds production and antioxidant enzymatic
activities^[47]6, [48]11–[49]13, the molecular basis of He-Ne laser
irradiation induced physiological changes for crops adapting to drought
environments is still poorly understood due to the absence of genomic
information.
A rapid and cost-effective approach to explore plant tolerance
mechanisms to abiotic stresses is to carry out transcriptomic analyses
using microarray-based or RNA-Seq-based technologies. More recently,
transcriptome sequencing has proven to be a powerful tool to discover
the molecular processes involved in plant response to various abiotic
stresses. Genome-scale transcriptome analyses have been employed on
diverse crops based on Illumina RNA-Seq to monitor gene expression in
response to heat stress^[50]14, cold stress^[51]15 and drought
stress^[52]16–[53]18. A recent genome-wide identification of
differentially expressed transcript derived fragments from water
deficit stressed root and leaf tissues in tetraploid cotton provided
their gene ontology, functional/biological distribution, and possible
roles of gene duplication^[54]19. Furthermore, Fracasso et al. compared
drought response of two sorghum genotypes characterized by contrasting
water use efficiency using high-throughput sequencing^[55]2. Although
much progress has been made in exploring the adaptive mechanisms of
plants to drought stress, genome-wide transcriptomic analysis of
drought stress tolerance conferred by laser irradiation in wheat has
not been reported. Using the high-throughput sequencing, we studied
He-Ne laser irradiation induced transcriptome changes in wheat
seedlings under drought stress, and believe it is important for
understanding of the molecular mechanism associated with laser
irradiation mediated drought tolerance in wheat.
Materials and Methods
Plant material and treatment conditions
Seeds of wheat (Triticum aestivum L. cv. Aikang No. 58, obtained from
Henan Academy of Agricultural Sciences) were surface sterilized for
3 min by immersion in 0.01% HgCl[2], soaked in distilled water for 24 h
prior to laser treatment, and then were air dried. A He-Ne laser
(wavelength 632.8 nm, power density 5.43 mW mm^−2, beam diameter 12 mm)
directly irradiated the embryo of wheat seeds for 3 min. Three
replications of 10 pure seeds were used for each of the different
treatment. One seed was pretreated only once by He-Ne laser
irradiation. The seeds were exposed to He-Ne laser one by one. The
He-Ne laser (Model No. MSHN5-A-B450MM) was made at Northwest University
(China). After He-Ne laser pretreatment, seeds were sown in plastic
pots (8 cm diameter at top, 6 cm diameter at bottom, and 8.0 cm height)
filled with a mixture of vermiculite and nutrient soil in 1:1 ratio and
irrigated with tap water. After germination, the seedlings were
cultured in a greenhouse under a 12 h photoperiod, 25/18 °C day/night
temperatures, light intensity of 400 μmol m^−2 s^−1, and relative
humidities of 60/75% (day/night). One-week-old seedlings (with one
fully expanded leaf) were randomly divided into four groups: seedlings
with no drought stress and no He-Ne laser irradiation were regarded as
the control group (CK), seedlings treated with natural drought stress
without water for 5 days (the soil relative water content was 50%, P),
seedlings pretreated with He-Ne laser irradiation alone (L), seedlings
pretreated with He-Ne laser irradiation before treated with natural
drought stress without water for 5 days (L + P). On the 5th day of
drought stress, leaves and roots were collected, respectively, and
immediately frozen in liquid nitrogen and stored at −80 °C until
further use.
Growth parameters
Growth parameters (plant height, root length, shoot dry weight and root
dry weight) were determined 5 days after He-Ne laser pretreatment or
drought stress. Relative water content (RWC) in leaves was calculated
according to the following formula^[56]20:
[MATH: RWC(%)=[(freshweight−dryweight)/(turgidweight−dryweight)]×100 :MATH]
Leaves of the different treated seedlings were collected and
immediately weighed (fresh weight, FW). They were rehydrated in water
for 24 h until fully turgid, surface dried, and reweighed (turgid
weight, TW). The tissues were then oven dried at 105 °C for 24 h and
re-weighed (dry weight, DW).
Determination of malondialdehyde (MDA), photosynthetic pigments and protein
concentration
Lipid peroxidation was assayed by measuring malondialdehyde (MDA)
content using the method described by Qiu et al.^[57]21. The content of
chlorophyll a, chlorophyll b, and total chlorophyll in wheat seedling
leaves were determined spectrophotometrically according to the method
of Lichtenthaler^[58]22. Soluble protein content in wheat seedlings
leaves was carried out according to the method described by Bradford
using bovine serum albumin as a standard^[59]23.
Total RNA extraction, libraries construction and illumina sequencing
Twenty individual wheat seedlings were pooled to create one treatment
and total RNA was extracted from different treatment using RNAsimple
total RNA kit (Tiangen, China) according to the manufacturer’s
instructions. The concentration and quality of total RNA in each sample
was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies,
Santa Clara, CA, USA). The library was constructed using the NEB Next
Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ispawich,
USA) following manufacturer’s instructions and four index codes were
added to attribute sequences to different samples. Briefly, mRNAs were
enriched from 3 ug total RNA of wheat tissues using magnetic beads with
Oligo (dT) (Life technologies, CA, USA), and then fragmented using
divalent cations under elevated temperature in the NEB proprietary
fragmentation buffer. Using these short fragments as templates, a
random hexamer primer was used to synthesize first-strand cDNA.
Second-stranded cDNAs were then synthesized using RNase H and DNA
polymerase I. These cDNA fragments were ligated with the adapters, and
these products were then purified and enriched with PCR to create the
final cDNA libraries. Finally, the library was sequenced by Novogene
Bioinformatics Technology Co., Ltd (Beijing, China) on an Illumina
HiSeq^TM 2000 platform. The clean reads were first obtained by removing
low-quality reads and reads containing adapters or poly-N stretches
from the raw data. Then, clean reads were mapped to our assembled wheat
transcriptome (SRR5119530) by using TopHat software (v2.0.12).
To maximize the number of genes included in wheat transcriptome, a cDNA
sample was prepared from an equal mixture of total RNA isolated from
different treatment wheat seedlings (with one fully expanded leaf), and
sequenced using the Illumina high-throughput sequencing platform. The
different treatment wheat seedlings were divided into four groups:
seedlings with no drought stress and no He-Ne laser irradiation,
seedlings treated with natural drought stress, seedlings were
pretreated with He-Ne laser irradiation alone, and seedlings pretreated
with He-Ne laser irradiation before treated with natural drought
stress. Raw sequence data are available in the NCBI’s Sequence Read
Archive (SRA) database with accession number SRR5119530. Generated
clean reads were aligned to the IWGSC wheat assembly
([60]http://archive.plants.ensembl.org/) and TGACv1 wheat assembly
([61]http://plants.ensembl.org/) using BWA and Bowtie2 software^[62]24.
The assembled wheat transcriptome contains 188,334 transcripts with a
mean length of 801 bp and 119,588 unigene with a mean length of
1,096 bp. The summary for the wheat transcriptome is shown in
Tables [63]S1 and [64]S2.
Differentially expressed genes (DEGs) detection and functional analysis of
DEGs
For gene expression analysis, the read numbers mapped to each gene were
counted using HTSeq v0.5.4p3 and then normalized to RPKM^[65]25 (reads
per kilobase per million mapped reads). Differential expression
analysis for each sample was performed using the DESeq R package. We
use false discovery rate (FDR) ≤ 0.05 and the absolute value of log2
(ratio) ≥ 1 as the threshold for judging the significance of
differentially expressed gene in different treatments. Gene Ontology
(GO) enrichment analysis of differentially expressed genes was
implemented using the GOseq R package and GO terms with q < 0.05 were
regarded as significantly enriched. Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathways analysis of the differential expression genes
was performed using the KOBAS software.
Quantitative real-time PCR
To validate the RNA-seq data, 9 genes were randomly selected to be
analyzed by qRT-PCR with a reference gene (Tubulin; Table [66]S3) and
specific primer pairs for selected genes were designed as shown in
Table [67]S3. Sample treatment and RNA isolation were obtained
following previously described above. The reverse-transcription
reactions were performed using the iScriptTM advanced cDNA Synthesis
Kit (Promega, WI, USA). Quantitative real time PCR was performed using
a Rotor-Gene 3000 real-time PCR detection system (Qiagen) with SYBR^®
qPCR Mix (Toyobo, Tokyo, Japan) and PCR amplifications were carried out
in 20 µl total volume reactions containing 2 µl diluted cDNA, 300 nM of
each primer, and 10 µl of the Thunderbird SYBR Green PCR Master Mix
with the following cycling conditions: 95 °C for 2 min, 35 cycles at
95 °C for 15 s, 60 °C for 15 s, and 72 °C for 20 s. Dissociation curve
analysis of amplification products was performed at the end of each PCR
to confirm that only one PCR product was amplified and detected. The
experiment was performed with at least three independent replicates,
and the comparative CT method (2^−ΔΔCt method) was used to analyze the
expression level of the different genes^[68]26.
Statistical analysis
The experiment was a completely random design with three replications,
and the data presented are means ± SEs. Each independent experiment was
a pooled sample from at least 20 wheat seedlings. Bars with different
letters above the columns in the figures indicate significant
differences at p < 0.05 by Duncan’s multiple range test.
Results
Effect of He-Ne laser pretreatment on growth parameters of wheat seedlings
under drought stress
Exposure to drought stress produced a significant growth inhibition in
wheat seedlings, as observed in plant height, root length, shoot dry
weight and root dry weight. Compared to the control (CK), drought
stress (P) caused a significant decrease (p < 0.05) in the plant
height, root length, shoot dry weight and root dry weight by 16.4,
19.19, 13.3 and 16.7 percent, respectively, in wheat seedlings. In
contrast, He-Ne laser pretreatment combined with drought stress (L + P)
caused an increase (p < 0.05) in the plant height, root length, shoot
dry weight and root dry weight by 21.8, 34.5, 11.5 and 25 percent,
respectively, compared with the seedlings treated with drought stress
alone (Table [69]1). Treatments with He-Ne laser alone (L) have little
effect on plant height, root length, shoot dry weight and root dry
weight in wheat seedlings compared with the control. These results
suggested that He-Ne laser pretreatment was responsible for the
enhancement of adaptive responses of wheat seedlings against drought
stress.
Table 1.
Effect of He-Ne laser pretreatment on root length, plant height, shoot
dry weight, and root dry weight of the 7-day-old wheat seedlings
treated with natural drought stress without water for 5 days.
CK P L L + P
Plant height (cm) 13.49 ± 1.34a 11.28 ± 1.31b 14.54 ± 1.01a
13.74 ± 1.01a
Root length (cm) 12.66 ± 2.31a 10.23 ± 1.89b 13.47 ± 1.85a
13.76 ± 1.10a
Shoot dry weight (g) 0.060 ± 0.005a 0.052 ± 0.003b 0.057 ± 0.003a
0.058 ± 0.004a
Root dry weight (g) 0.024 ± 0.003a 0.020 ± 0.004b 0.024 ± 0.002a
0.025 ± 0.002a
[70]Open in a new tab
Different treatment represents: The control (CK), natural drought
stress (P), 3 min laser radiation (L), 3 min laser radiation + natural
drought stress (L + P). Data are means of 30 seedlings, and each mean
in a line followed by a different letter indicates that there are
significant differences at 0.05 level according to Duncan’s multiple
range test.
Effect of He-Ne laser pretreatment on drought stress tolerance in wheat
seedlings
To assess oxidative stress damage generated by drought stress, MDA
content as an important indicator of lipid peroxidation was measured.
Drought stress alone for 5 days caused 17.3% increase (p < 0.05) in MDA
content compared with the control (Fig. [71]1). On the contrary, MDA
content was decreased significantly (p < 0.05) in wheat seedlings
pretreated with He-Ne laser under drought stress compared with drought
stress alone. Figure [72]1 also showed that treatments with He-Ne laser
alone dramatically decreased the concentration of MDA compared with the
control. These results indicated that He-Ne laser pretreatment could
alleviate drought stress induced oxidative damage in wheat seedlings.
Figure 1.
Figure 1
[73]Open in a new tab
Effect of He-Ne laser pretreatment on the MDA, protein, photosynthetic
pigments concentration and the relative water content of the 7-day old
seedlings treated with drought stress for 5 days. See notes to
Table [74]1. Bars are means ± standard error of 6 replicates. Different
letters above bars indicate significant difference at the 0.05
significant level according to Duncan’s multiple range test.
As shown in Fig. [75]1, a remarkable reduction in relative water
content (RWC) (12.5%), protein content (71.4%), chlorophyll a (38.7%),
chlorophyll b (42.1%), and chlorophyll (a + b) (42.2%) content were
noticed in drought stress treated wheat seedlings, suggesting that
drought stress negatively affected various aspects of wheat seedlings
at physiological level. However, He-Ne laser pretreatment combined with
drought stress (L + P) significantly improved relative water content,
protein and photosynthetic pigments concentration compared to drought
stress alone (Fig. [76]1). Treatments with He-Ne laser alone (L) had no
significant effect on relative water content as compared to the
control. These results demonstrated that He-Ne laser pretreatment could
improve drought stress tolerance in wheat seedlings.
An overview of transcriptome sequencing datasets
To further elucidate the underlying molecular basis of He-Ne laser
irradiation induced drought tolerance, RNA-Seq was employed to
investigate the changes of genome-wide gene expression in wheat
seedlings pretreated with He-Ne laser irradiation whether or not the
seedlings were subjected to drought stress. Approximately 24.96–35.13
million 100 bp paired-end clean reads obtained for the control, drought
stress (P), 3 min laser radiation (L) and 3 min laser
radiation + drought stress, respectively were generated after adapter
trimming and filtering low-quality reads (Table [77]2). The average
Q20, Q30, and GC contents were 95.96%, 89.22%, and 55.11%, respectively
and the clean reads of Q20 occupied over than 95% of the total,
suggesting high quality sequencing. Then, we used TopHat v2.0.9
software to map all clean reads to the wheat transcriptome. The reads
mapping to the reference transcriptome were categorized into two
classes: uniquely mapped reads, that are reads that map to only one
position in the reference transcriptome, and multi-position match, that
are reads mapping to more than one position in the reference
transcriptome (Table [78]2). Of the total clean reads from the four
sample groups, 65.82–67.65% were uniquely mapped, and 4.75–6.14% were
mapped to multiple loci (Table [79]2). It has been found that
70.6–73.8% of the reads were mapped to the reference transcriptome.
Major mapping reads indicated reliable transcriptome data.
Table 2.
Summary of RNA-seq data and reads mapping.
Sample name CK P L L + P
Raw reads 30,890,152 36,407,862 31,799,704 26,286,622
Clean reads 29,987,786 35,133,470 30,566,204 24,9552,36
Clean bases 3.75 G 4.39 G 3.89 G 3.12 G
Q20 (%) 95.15 95.45 95.47 95.3
Q30 (%) 88.82 89.4 89.41 89.12
GC (%) 54.58 55.82 56.15 56.37
Total mapped 19,960,572 (75.56%) 22,695,390 (74.60%) 19,701,309
(74.45%) 15,616,658 (72.58%)
Multiple mapped 2,440,188 (6.14%) 2,442,640 (4.95%) 2,079,018 (4.80%)
1,685,476 (4.75%)
Uniquely mapped 17,220,384 (67.42%) 20,252,750 (67.65%) 17,622,291
(67.65%) 13,931,182 (65.82%)
[80]Open in a new tab
Different treatment represents: The control (CK), natural drought
stress (P), 3 min laser radiation (L), 3 min laser radiation + natural
drought stress (L + P).
Effect of He-Ne laser pretreatment on differentially expressed gene in wheat
seedlings under drought stress
In this study, RNA-Seq analysis was performed to obtain a comprehensive
view of gene expression profile in He-Ne laser pretreated wheat
seedlings under drought stress. Hierarchical clustering of
differentially expressed genes demonstrated that differential gene
expression occurred in He-Ne laser pretreated wheat seedlings under
drought stress (Fig. [81]2A). The expression of each gene was
normalized to the number of reads per kilobase per million clean reads
(RPKM) to compare among different samples. We then used the DESeq R
package to identify differentially expressed genes (DEGs). Using false
discovery rate (FDR) ≤ 0.05 and fold change ≥1 as the significance
cutoff, the expression of 338 DEGs was found to be significantly
regulated between drought stress and CK libraries, including 182 and
156 genes that were up- and down-regulated, respectively. The 410 DEGs
in both the He-Ne laser alone treatment and CK libraries showed
quantitative differences. In addition, we compared L + P and P
libraries, and 820 variant genes were found, of which 442 were
up-regulated and 378 were down-regulated (Fig. [82]2B). A Venn diagram
showed the distribution of differentially expressed genes from He-Ne
laser pretreatment on wheat seedlings under drought stress in
Fig. [83]S1. Interestingly, larger numbers of genes were up- or
down-regulated at L + P than P, implying that the He-Ne laser
pretreatment could induce more complicated transcript regulation in
wheat seedlings under drought stress.
Figure 2.
Figure 2
[84]Open in a new tab
Transcriptome analysis of differentially expressed genes in He-Ne laser
pretreated wheat seedlings under drought stress. See notes to
Table [85]1. (A) Hierarchical clustering of all the DEGs based on
log[10] RPKM (number of reads per kilobase per million clean reads)
values. The color (from red to blue) represents gene expression level
from high to low. (B) Changes in differentially expressed genes (DEG)
in He-Ne laser pretreated wheat seedlings under drought stress. The
number of up- and down-regulated genes between P and CK, L and CK, and
L + P and P are summarized.
Functional characterization of differentially expressed genes in He-Ne laser
pretreated wheat seedlings under drought stress
To further investigate the main biological functions of differentially
expressed genes in He-Ne laser pretreatment on wheat seedlings under
drought stress, gene ontology (GO) analysis were used to classify the
functions of the differentially expressed genes. Based on sequence
homology, 1,568 DEGs could be categorized into cellular component,
molecular function, and biological process, in which there are 7, 12
and 15 functional groups, respectively (Fig. [86]3). Among these
groups, the terms “membrane”, “hydrolase activity”, and “metabolic
process” are dominant in each of the three main categories,
respectively. Notably, some genes associated with “response to
oxidative stress”, “catalytic activity”, “antioxidant activity”, and
“oxidation-reduction” were enriched in He-Ne laser pretreatment
combined with drought stress, suggesting that He-Ne laser pretreatment
induced antioxidant response was activated in wheat seedlings under
drought stress.
Figure 3.
Figure 3
[87]Open in a new tab
GO functional analysis of differentially expressed genes (DEGs) in P vs
CK and L + P vs P. See notes to Table [88]1. (A) Biological process.
(B) Molecular function. (C) Cellular component.
To further identify metabolic or signal transduction pathways in which
the DEGs are likely to be involved in drought stress tolerance
conferred by He-Ne laser irradiation in wheat, pathway enrichment
analysis was performed using KEGG database
([89]http://www.genome.ad.jp/kegg/). Among DEGs of He-Ne laser
pretreated wheat seedlings combined with drought stress (L + P) and
drought stress alone (P) treatments tested the following 6 pathways,
with a KEGG pathway annotation, were significantly affected: carbon
fixation in photosynthetic organisms, photosynthesis-antenna proteins,
plant pathogen interaction, pyruvate metabolism, photosynthesis and
linoleic acid metabolism pathways (q < 0.05; Fig. [90]4).
Figure 4.
Figure 4
[91]Open in a new tab
KEGG pathway enrichment analysis of DEGs between P and L + P treatment.
The left Y-axis shows the KEGG pathway. The X-axis shows the Rich
factor. The enrichment factor indicates the ratio of differential
expression unigenes enriched in this pathway to the total number of
annotated unigenes. The size and color of each point represents the
number of genes enriched in a particular pathway and the q-values. A
larger enrichment factor value and lower q-values indicates a greater
degree of enrichment.
Differentially expressed genes by He-Ne laser pretreatment involved in
drought stress tolerance in wheat seedlings
Drought stress changes gene expression in He-Ne laser pretreated wheat
seedlings, and several genes expression level exhibited highly dynamic
changes (|log[2]Ratio| ≥ 2, Fig. [92]5), including genes encoding WRKY
transcription factors, proteins kinase, stress-associated proteins
(plant disease resistance response protein, class III peroxidase,
catalase immune-responsive domain, superoxide dismutase, glutathione
S-transferase), photosynthesis-associated proteins (photosystem II PsbR
protein, chlorophyll a/b binding protein, ATP synthase),
transporter-associated proteins (ABC transporter, peptide transporter,
drug/metabolite transporter, amino acid transporter, sugar/inositol
transporter) (Fig. [93]5 and Table [94]S4). Interestingly, transcripts
of some genes involved in drought stress tolerance accumulated to
higher level in He-Ne laser pretreated wheat seedlings than drought
stress alone, suggesting that these genes might contribute to the
enhanced drought stress tolerance in He-Ne laser pretreated wheat
seedlings.
Figure 5.
Figure 5
[95]Open in a new tab
Expression profile of 40 differentially expressed genes involved in
transcription regulation, photosynthesis, transport, defense/stress
response, and others in He-Ne laser pretreated wheat seedlings under
drought stress. The bar represents the scale of the expression levels
for each gene (log[10]RPKM (number of reads per kilobase per million
clean reads)) in the different treatments as indicated by red/green
rectangles. Genes in red indicate up-regulation and in green indicate
down-regulation. All genes in this list have a P-value for differential
expression <10^−5. The control (CK), natural drought stress (P), 3 min
laser radiation (L), 3 min laser radiation + natural drought stress
(L + P). Complete information for each gene list can be found in
Table [96]S4.
Quantitative real-time-PCR validation of differentially expressed transcripts
from RNA-Seq
To further validate the reliability of our Illumina sequencing
analyses, we have nine differentially expressed genes by quantitative
real time RT-PCR (qRT-PCR) using the independently collected samples
that were in different treatments as those used for RNA-Seq, including
genes encoding plant peroxidase, class III peroxidase, glutathione
S-transferase, catalase immune-responsive domain, heat shock protein 70
family, WRKY transcription factor, glycoside hydrolase, photosystem II
PsbR protein, drug/metabolite transporter, respectively. The results
presented in Fig. [97]6 showed that the expression levels of nine genes
were higher in He-Ne laser pretreatment combined with drought stress
than drought stress alone. The expression trends of these genes agreed
with the RNA-Seq data (Fig. [98]6), and a significant correlation
between the expression changes (fold difference) measured by RNA-Seq
and those by qRT-PCR (Fig. [99]6), indicating the reliability of our
sequencing data as well as further confirming the He-Ne laser
pretreatment conferred improved drought stress tolerance in wheat
seedlings.
Figure 6.
Figure 6
[100]Open in a new tab
Expression of nine differentially expressed genes in He-Ne laser
pretreatment on wheat seedlings under drought stress. Correlation
between the expression profiles of the nine transcripts determined by
RNA-Seq and qRT-PCR. See notes to Table [101]1. Genes are (a) plant
peroxidase; (b) class III peroxidase; (c) glutathione S-transferase;
(d) catalase immune-responsive domain; (e) heat shock protein 70
family; (f) WRKY transcription factor; (g) glycoside hydrolase; (h)
photosystem II PsbR protein; (i) drug/metabolite transporter. The
y-axes show expression levels (fold difference) determined by Inline
graphic qRT-PCR and Inline graphic RNA-Seq.
Discussion
He-Ne laser pretreatment on drought tolerance as indicated by morphological
and physiological changes
Drought stress adversely influences many physiological processes of
wheat, limits wheat growth, and decreases wheat productivity^[102]27,
[103]28. Results of the current study further confirmed the negative
effect of drought stress on plant growth. In this study, our results
showed that drought stress significantly inhibited plant height, shoot
dry weight, root length and root dry weight, relative water content
(RWC), and protein content in wheat seedlings. However, He-Ne laser
pretreatment remarkably promoted wheat seedlings growth parameters such
as plant height, shoot dry weight, root length and root dry weight,
RWC, and protein content under drought stress. The morphological and
physiological responses of wheat seedlings suggest that He-Ne laser
pretreatment could improve drought stress tolerance in wheat seedlings,
in accordance with the enhanced resistance to cold stress^[104]7, UV-B
radiation^[105]12, [106]13, cadmium stress^[107]10, and salt
stress^[108]11. However, the mechanism of how He-Ne laser alleviated
the negative effect of drought stress in wheat seedlings need to be
further addressed.
Potential DEGs playing critical roles in drought tolerance conferred by He-Ne
laser irradiation in wheat
Drought stress triggers significant molecular and physiological changes
in plants, and a global transcriptional reprogramming is considered as
the important molecular response of plants to adapt/cope with drought
stress. To gain insight into the molecular response of He-Ne laser
enhanced drought stress tolerance, we performed RNA-Seq analysis and
investigated transcriptional differences in He-Ne laser pretreated
wheat seedlings under drought stress. A total of 820 genes (442
up-regulated and 378 down-regulated) were found differentially
expressed in He-Ne laser pretreated wheat seedlings under drought
stress (L + P) with respective to drought stress alone (P). In
addition, we compared P and CK libraries, and 338 variant genes were
found, of which 182 were up-regulated and 156 were down-regulated.
These results indicated considerable changes of gene expression in
He-Ne laser pretreated wheat seedlings under drought stress.
Furthermore, functional categories of the DEGs by GO term enrichment
analysis in our present study showed that transcripts that function in
regulation of photosynthesis and antioxidant defense response were
expressed predominantly in He-Ne laser pretreated wheat seedlings. An
increasing number of transcriptome studies conducted in diverse plant
species have also noted the enrichment of GO terms and metabolic
pathways related to antioxidant defense response^[109]29, [110]30.
Fracasso et al. demonstrated that differentially expressed transcripts
in photosynthesis, antioxidant and secondary metabolism might play
essential roles in tolerant sorghum genotypes adaptation to drought
stress^[111]2. In addition, a large number of genes belonging to
various metabolic pathways, such as photosynthesis and carbohydrate
metabolism, abiotic stress, regulation of transcription were
significantly differentially expressed in He-Ne laser pretreated wheat
seedlings, leading us to conclude that most of the genes we identified
are involved in response and adaptation to drought stress in wheat.
He-Ne laser pretreatment enhances photosynthesis by increasing the
transcripts of photosynthesis-related genes
Photosynthesis plays a central role as an energy source for plant
metabolism^[112]13, [113]31. Drought stress inhibits photosynthetic
activity in plants by changing chlorophyll content, and a decrease of
chlorophyll content implies lowered light harvesting, thus leading to
overall growth retardation^[114]27, [115]31. In the present study, we
found that drought stress significantly decreased chlorophyll content
in wheat seedlings, but chlorophyll content was reversed back to a
similar level as the control in wheat seedlings pretreated with He-Ne
laser. In agreement with these results, Chen et al. demonstrated that
He-Ne laser treatment of wheat under UV-B radiation significantly
increased chlorophyll content and photosynthetic electron transport
chain efficiency^[116]13, thus resulting in the increase of
photosynthetic activities. The increased chlorophyll content of wheat
seedlings in He-Ne laser pretreatment may reflect changes in expression
levels of transcripts regulating photosynthetic activity. Indeed, five
genes encoding proteins known to regulate photosynthetic activity were
found to be up-regulated in He-Ne laser pretreated wheat seedlings
under drought stress. Recently, a number of genes have been reported to
be able to regulate drought tolerance through improving the
photosynthetic capacity. By using high-throughput sequencing, Zhang et
al. indicated that an increased expression of a large number of genes
related to photosynthesis pathway in upland rice could maintain
relatively higher photosynthesis activity under drought
conditions^[117]31, which might result in drought stress adaptation. In
this study, He-Ne laser induced the upregulation of most
photosynthesis-related genes, involving in PSII PsbR protein,
chlorophyll a/b binding protein and ATP synthase. Furthermore, qRT-PCR
from independently generated samples verified that the gene encoding
PsbR protein had significantly higher expression in the He-Ne laser
pretreated wheat seedlings under drought stress. These results suggest
that He-Ne laser irradiation could enhance drought stress tolerance in
wheat seedlings by improving photosynthesis at transcriptomic and
physiological level.
Genes encoding transcription factors are important for He-Ne laser pretreated
wheat seedlings in response to drought stress
Transcription factors (TFs) are important regulators of gene expression
and are known to play crucial roles in various biological processes
including responses to drought stress^[118]32–[119]34. TFs belonging to
MYB, AP2/ERF, bHLH, and WRKY families have been well characterized for
their regulatory roles in the response of plants to abiotic
stress^[120]35, [121]36. There have been a number of studies
demonstrating that transgenic plants overexpressing genes encoding TFs
can greatly enhance their tolerance to various abiotic stresses such as
salinity, cold, and drought^[122]37, [123]38. The expression patterns
of WRKY family were investigated in both the root and leaf of bread
wheat under drought conditions. Through in silico searches, 35 bread
wheat WRKY genes were detected by drought stress and the expression of
most of them was known to be down-regulated by drought stress
treatment^[124]39. Furthermore, overexpression of wheat WRKY genes
could enhance drought tolerance in transgenic tobacco^[125]38. More
recently, several studies also revealed that R2R3-MYB TFs in
Arabidopsis (AtMYB44, AtMYB60, and AtMYB61) are involved in regulation
of stomatal aperture in response to drought stress^[126]40. Our RNA-Seq
data indicated that these genes encoding WRKY, bHLH, and R2R3-MYB TFs
were up-regulated in He-Ne laser pretreated wheat seedlings under
drought stress, suggesting that these TFs might contribute to the
enhanced drought stress tolerance in He-Ne laser pretreated wheat
seedlings.
ROS-scavenging systems are important for He-Ne laser pretreated wheat
seedlings in response to drought stress
Drought stress will inevitably result in a massive production of
reactive oxygen species (ROS), which can cause lipid peroxidation and
leads to oxidative destruction of many cellular structures and
components^[127]41–[128]43. In order to avoid the oxidative damage,
plants have developed a complex antioxidative defense system to cope
with the stress induced oxidative damages. Many antioxidant enzymes,
such as superoxide dismutase (SOD), peroxidases (POD), catalases (CAT)
and glutathione S-transferase (GST) play a crucial role in scavenging
elevated levels of ROS, thus protecting cells from oxidative damage in
plants^[129]21, [130]42, [131]43. It was recently reported that
elevated antioxidant levels are associated with tolerance to abiotic
stress^[132]44. Our previous study demonstrated that He-Ne laser
pretreatment significantly increased the activities of antioxidant
enzymes (SOD, POD, APX and CAT) involved in ROS-scavenging in leaves,
and thus enhanced antioxidant ability in wheat seedlings under drought
stress^[133]8, [134]9. In the present study, the expression of several
genes encoding plant peroxidase, class III peroxidase, glutathione
S-transferase and catalase were up-regulated in He-Ne laser pretreated
wheat seedlings with respect to drought stress alone (Fig. [135]6). Our
results are supported by the results of Fracasso et al., who suggested
that an increased expression of a large number of genes involved in
antioxidant system has been associated with decreased oxidative damage
and enhanced stress tolerance in different drought tolerant sorghum
genotypes^[136]2. Using qRT-PCR, Gao et al. have also shown that He-Ne
laser pretreatment can enhance antioxidant defense systems and improve
salt stress tolerance by increasing the gene expression levels of SOD,
POD and CAT, which were in accordance with the increase of SOD, POD,
and CAT activity^[137]11. Taken together, our results suggest that the
enhanced gene expression of SOD, POD, CAT and GST might contribute to
the increases in the activities of SOD, POD, CAT and GST in He-Ne laser
pretreated wheat seedlings, thus resulting in the protection against
oxidative damage induced by drought stress.
Genes related to solute transport are important for He-Ne laser pretreated
wheat seedlings in response to drought stress
Transport processes are very important in the mobilization and
accumulation of solutes. Furthermore, these processes play an important
role in cell detoxification pathways during adaptation to drought. An
increasing number of transporters have been recognized to participate
in a multitude of physiological processes that allow the plant to adapt
to changing environments and to cope with biotic and abiotic stresses,
as well as detoxification processes^[138]45, [139]46. Wang et al. have
demonstrated that several transport-associated unigenes were
exclusively detected in Pyropia haitanensis in response to
rehydration^[140]45, which encode amino acid transporters, ion
transporters, sugar/inositol transporter, and peptide transporter. Many
amino acid transporters (AATs) genes are known to function in
mitigating water stress conditions in plants, especially by
facilitating the transport of stress-related compounds and compatible
solutes, such as proline, betaine, and a variety of
carbohydrates^[141]46. Furthermore, over-expression of sucrose
transporters (SUTs) has been correlated with improved drought
tolerance^[142]47. Our results indicated that the expression level of
genes related to the transport of amino acids and other solutes,
including malate, inositol, and sugar were significantly up-regulated
in He-Ne laser pretreated wheat seedlings in response to drought
stress. The differential expression of these transporters suggested
that the uptake and translocation of various nutrients, like sugar,
malate, inositol, could be affected by He-Ne laser irradiation.
Therefore, it is likely that during water deficit, the upregulation of
unigenes encoding various amino acids transporters, peptide
transporters, luminum-activated malate transporter and sugar/inositol
transporter in He-Ne laser pretreated wheat seedlings could contribute
to import of the nutrients and export of secondary metabolites and
other toxic compounds that protect wheat seedlings from drought stress.
In conclusion, we analyzed the gene expression profiles of laser
irradiation mediated drought-stress tolerance in wheat using RNA-Seq
analysis in the present study. Our results showed that 338 genes (182
up-regulated and 156 down-regulated) were differentially expressed in
wheat seedlings under drought stress (P) when compared with the control
(CK). However, He-Ne laser irradiation induced 820 differently
expressed genes, including 442 up-regulated and 378 down-regulated DEGs
in wheat seedlings drought stress with respect to drought stress alone,
indicating considerable changes of gene expression in He-Ne laser
pretreated wheat seedlings under drought stress. Genome-wide
transcriptomic analysis presented in this study has expanded our
knowledge of this process by identifying significantly altered genes
involved in photosynthesis, nutrient uptake and transport, homeostasis
control of reactive oxygen species and transcriptional regulation. To
the best of our knowledge, this study is the first to characterize
potential roles of laser irradiation mediated drought-stress tolerance
in wheat at the transcriptional level.
Electronic supplementary material
[143]Supplementary Information^ (191.2KB, pdf)
Acknowledgements