Comparative transcriptome analysis of the Asteraceae halophyte Karelinia caspica under salt stress
BMC Research Notes
Comparative transcriptome analysis of the Asteraceae halophyte Karelinia caspica under salt stress
Xia Zhang 0
Maoseng Liao 0
Dan Chang 0
Fuchun Zhang 0
0 Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University , 14 Sheng li Road, Urumqi 830046 , China
Background: Much attention has been given to the potential of halophytes as sources of tolerance traits for introduction into cereals. However, a great deal remains unknown about the diverse mechanisms employed by halophytes to cope with salinity. To characterize salt tolerance mechanisms underlying Karelinia caspica, an Asteraceae halophyte, we performed Large-scale transcriptomic analysis using a high-throughput Illumina sequencing platform. Comparative gene expression analysis was performed to correlate the effects of salt stress and ABA regulation at the molecular level. Results: Total sequence reads generated by pyrosequencing were assembled into 287,185 non-redundant transcripts with an average length of 652 bp. Using the BLAST function in the Swiss-Prot, NCBI nr, GO, KEGG, and KOG databases, a total of 216,416 coding sequences associated with known proteins were annotated. Among these, 35,533 unigenes were classified into 69 gene ontology categories, and 18,378 unigenes were classified into 202 known pathways. Based on the fold changes observed when comparing the salt stress and control samples, 60,127 unigenes were differentially expressed, with 38,122 and 22,005 up- and down-regulated, respectively. Several of the differentially expressed genes are known to be involved in the signaling pathway of the plant hormone ABA, including ABA metabolism, transport, and sensing as well as the ABA signaling cascade. Conclusions: Transcriptome profiling of K. caspica contribute to a comprehensive understanding of K. caspica at the molecular level. Moreover, the global survey of differentially expressed genes in this species under salt stress and analyses of the effects of salt stress and ABA regulation will contribute to the identification and characterization of genes and molecular mechanisms underlying salt stress responses in Asteraceae plants.
Salinity is a major environmental factor limiting plant
growth and productivity. Thus, salt-tolerant halophytes
serve as an excellent resource for the identification of
desirable traits and the subsequent development of new crop
]. Understanding the salt tolerance mechanisms
in such plants represents an important step towards
generating crop varieties capable of coping with
environmental stresses. Towards this end, there is still much to learn
about the diverse mechanisms employed by halophytes to
cope with salinity stress.
Karelinia caspica, an herbaceous Asteraceae perennial
that grows in saline deserts and swamps [
broadspectrum resistance to pests and is also tolerant to salinity,
drought, low temperatures, and high temperatures [
to its extreme desalination capacity, K. caspica is viewed as
a good pioneer plant for the improvement of saline soil [
As a secretohalophyte, K. caspica actively absorbs and
discharges salt through special glands and salt holes on the
leaf surface [
]. At the same time, salt stress promotes K.
caspica succulence , which is typical of euhalophytes.
Considering these mechanisms in aggregate, the salt
tolerance of K. caspica as intermediate between that of
euhalophytes and recretohalophytes is worth examining.
For most non-model organisms, the lack of whole-genome
sequencing data continues to represent a major hurdle for
research, as genome sequencing is still largely impractical for
most eukaryotes and cannot be finished in a short timeframe.
In contrast, high-throughput transcriptome analysis can be
performed whether or not genomic sequences for the
organism of interest are available [
] and thus represents a feasible
approach for organisms that have not been sequenced. To
date, transcriptome analysis has been employed in a wide
range of eukaryotes including the following: yeast [
], humans [
], Arabidopsis [
], Caenorhabditis elegans [
], and Vitis vinifera [
In addition to regulating diverse aspects of plant growth
and development, abscisic acid (ABA) is also required for
plant resistance to drought and salt stress [
Numerous studies have aimed to elaborate the cellular and
molecular responses of plants to ABA, including those related
to ABA sensing, signaling, metabolism, and transport.
To globally survey the salinity-induced ABA responses in
K. caspica, RNA-Seq technology was used. The
transcription profiles of control and salt-stressed plants were
compared, and dynamic changes in the transcriptome were
analyzed. The goal of the present work was to elucidate
physiological processes, including those involved in the
ABA regulatory network, that are induced by salt stress in
the halophyte K. caspica at the transcriptomic level.
The K. caspica seeds used in this study were obtained
from saline land (Wujiaqu, Xinjiang, China). Specific
permission was not required for this collection activity
at these locations, which are considered wastelands, and
the field studies did not involve endangered or protected
species. Eight-week-old seedlings germinated in perlite:
vermiculite substrate (3:1) supplemented with 300 mM
NaCl at 25°C and grown under a 16-hour photoperiod
were used for the expression analysis. The treated and
nontreated groups were sampled at 3, 6, 12, 24, 36, and 48 hours.
After washing with deionized distilled water, the material
from the different time points was combined to generate the
final samples used for cDNA library preparation.
Preparation of cDNA Libraries for RNA-Seq
For each treatment (i.e., the control and salt stress
treatments), approximately 100 g fresh material was used for
RNA preparation. Total RNA was extracted using the
RNAqueous Kit [
] then subsequently treated with RNase-free
DNase I (QIAGEN #79254) to remove residual genomic
DNA. For each treatment, mRNAs were treated with Truseq
RNA sample preparation kit (Illumina-15026495, USA),
firstly purified from the 20 mg total RNA using oligo (dT)
magnetic beads and fragmented using fragmentation buffer.
Cleaved short RNA fragments were used for first-strand
cDNA synthesis using reverse transcriptase and hexamer
primer, followed by second strand cDNA synthesis using
DNA polymerase I and RNase H. Following a quality check
using an Agilent 2100 Bioanalyzer, the cDNA libraries were
used for sequence analysis using the Illumina HiSeq TM
Raw sequencing data were deposited in the DDBJ/EMBL/
GenBank database (accession number GANI00000000).
After the adapter sequences were trimmed from the raw
reads, empty reads and reads containing unknown
nucleotides (Ns) > 5 were removed. The remaining clean reads
were de novo assembled into unigenes using Trinity
software (trinityrnaseq_r2012-06-08 edition). The paired-end
method was then used to acquire a single set of
nonredundant unigenes. Glimmer 3.02 and EMBOSS_6.3
software were used to analyze the coding sequences (CDS) of
the unigenes. All non-redundant unigenes were used to
perform BLAST searches and to obtain annotation
information in the following databases: NCBI nr, Swiss-Prot,
Kyoto Encyclopedia of Genes and Genomes (KEGG), and
Cluster of Orthologous Groups (KOG). WEGO [
used to classify GO function, which was analysis using the
Blast2go program [
Identification of differentially expressed genes
RPKM (reads per kilobase per million reads) values were
used to evaluate expression and to quantify transcript
], and differentially expressed unigenes [
identified by fold change values and fisher test. In the
present study, the fold.change (AB) value ≥ 1 were
choosen was used to as the threshold to determine significant
differences in gene expression. For the pathway
enrichment analysis, all differentially expressed unigenes were
mapped to terms in the KEGG database. A search was
then performed for significantly enriched KEGG terms
compared to the whole transcriptome background.
Quantitative RT-PCR (qRT-PCR) analysis
For qRT-PCR, total RNA was isolated from 200 mg frozen
materials as described above. Three biological replicates
were performed for both the control and salt stress-treated
seedlings. For each qRT-PCR reaction, 100 ng of RNA that
had been treated to remove genomic DNA was used as
template for cDNA synthesis. Additional file 1: Table S1
lists the sequences of the primer pairs used for qRT-PCR.
The relative transcript levels of selected genes were
quantified using Platinum® SYBR® Green qPCR SuperMix-UDG
(Invitrogen, CA, USA). The reactions were performed using
the 7500 Real-Time PCR system and software (Applied
Biosystems, CA, USA). KcACTIN, which was constant under
the tested conditions, was included for normalization in
each qRT-PCR run. Average expression ratios (PQ/C)
were calculated using the ΔΔCT method, and log2
fold-change values were used.
Results and discussion
Raw reads processing and assembly
To obtain transcript information for K. caspica, two
cDNA libraries were prepared for the control and salt
stress-treated plants then sequenced using the Illumina
sequencing platform. The raw reads were transformed by
base calling from the image data output from sequencing.
After trimming the adapter sequences and removing
sequences with unknown or low quality bases,
approximately 50.35 and 51.58 million clean reads were obtained
for the control and salt stress samples, respectively.
Trinity software (trinityrnaseq_r2012-06-08) and the
pairedend method were used for de novo assembly of the clean
reads into contigs. A total of 287,185 transcripts with
different lengths were generated from both of the treatments
(Additional file 2: Table S2), which provided abundant
information for subsequent analysis of salt stress-associated
genes in K. caspica.
Functional annotation and GO assignments of the assembled transcripts
The unigene sets obtained from the K. caspica
transcriptome data were annotated based on protein sequence
homology. First, we used glimmer3.02 and EMBOSS_6.3.1
software to transform all transcripts with sequences longer
than 100 bp into reliable coding sequences (CDS). The
320,260 CDS produced were used for further annotation
with the NCBI nr, Swiss-Prot, TrEMBL, CDD, pfam, and
KOG databases. From this analysis, 216,415 unigenes were
identified that exhibited high sequence similarity with
known gene sequences (Additional file 3: Table S3 and
Additional file 4: Table S4). Subsequently, a total of 18,378
sequences were assigned to 25 KOG (clusters of
orthologous groups for eukaryotic complete genomes) terms.
Among the terms identified, the three highest represented
categories were as follows: signal transduction mechanisms
(4,561); posttranslational modification, protein turnover,
chaperones (3,441); and general function prediction (3,271)
(Figure 1). All of the unigenes were queried in the GO
database (Gene Ontology, an international standardized gene
functional classification) to classify their predicted
functions. From this analysis, 35,533 unigenes were grouped
into three functional categories, “biological process”,
“cellular component”, and “molecular function”, with the subsets
of sequences further divided into 33, 18, and 17
subcategories in these three groups, respectively. The largest
subcategory in the “biological process” group was “metabolic
process”, which included 12.1% of the unigenes in the
subcategory. In the “cellular component” and “molecular
function” categories, “cell” and “binding activity” were the most
abundant GO terms, representing 7.5% and 11.3% of each
subcategory, respectively. In addition, there were high
percentages of unigenes in the “cell part”, “catalytic activity”,
and “cellular process” categories and only a few unigenes in
the “receptor regulator activity”, “locomotion”, “cell
junction”, and “symplast” categories (Figure 2).
Changes in gene expression under salt stress
To investigate the molecular response to salt stress
exposure, unigene transcript levels in the control and
salt stress treatments were calculated as RPKM (reads per
kilobase per million reads), which eliminates the influence
of gene length and sequencing discrepancy in calculating
gene transcript levels and permits a direct comparison
between treatments [
]. Based on the RPKM values
observed, 60,127 differentially expressed unigenes were
detected (Additional file 5: Table S5), with 38,123 and
22,005 up- and down-regulated genes, respectively. Salinity
imposes a water deficit and ion stress, which have
wideranging effects on the activity of plant cells, including
inhibition of essential enzymes, cell membrane destabilization, a
decrease in nutrient supply, and overproduction of reactive
oxygen species (ROS) [
]. The extensive variation
observed in the transcriptome (67.3%) indicates complex
transcriptional changes in K. caspica and comprehensive
salt-stress influence on the cellular activity of K. caspica.
Functional annotation of differentially expressed unigenes under salt stress
To identify unigenes involved in metabolic or signal
transduction pathways that were significantly enriched
under salt stress, all of the differentially expressed
sequences were queried in the KEGG database and
compared to whole transcriptome data. Among the 60,127
differentially expressed transcripts (DETs), 13,848 genes
were well annotated, while the remaining 46,239 genes
had low sequence similarity to known sequences in the
current database and therefore represent potentially
novel salt-stress responsive genes. The potentially large
number of unknown regulated genes suggested that
factors involved in salt stress responses in Asteraceae may
be distantly related to those identified in other genera.
Pathway enrichment analysis revealed that many genes
for which annotation data were available were directly or
indirectly involved in the salt stress response, namely,
primary metabolism, cellular processes, plant hormone
signal transduction, plant-pathogen interaction,
biosynthesis of secondary metabolism, and plant circadian
rhythm (Figure 3 and Additional file 6: Table S6). These
findings underscore the large scale re-coordination that
occurs during short-term acclimation to salt stress
exposure. Among the 4,023 DETs with pathway
annotation, 172 DETs were found to be involved in the plant
hormone signal transduction after salinity exposure, and
68 DETs were associated with MAPK signaling pathway
(Table 1). Since very little information about the signaling
cascades and the pathway of salinity sensing in Asteraceae
is available, the presently identified sequences provide
important clues for screening these putative salt stress
responsive genes and their associated genes.
Functional annotation of differentially expressed unigenes involved in ABA-signaling under salt stress
ABA plays a key role in a wide range of developmental
processes and adaptive stress responses to environmental
stimuli in plants [
]. Many studies have focused on the
cellular and molecular responses in plants to ABA, including
ABA metabolism, transport, sensing, and signaling. We
therefore focused on these physiological processes and
highlighted potentially informative findings determined from
ABA is synthesized from carotenoids. 9-cis-epoxycarotenoid
dioxygenase (NCED) breaks down the 11, 12 double bond
of 9-cis violaxanthin, which results in the formation of
C15 xanthoxin within plastids. The subsequent steps of
xanthoxin conversion to abscisic aldehyde are catalyzed
by abscisic-aldehyde oxidase (AAO3) and xanthoxin
dehydrogenase (ABA2) in the cytosol [
]. In the
saltstressed K. caspica transcriptome, NCED (comp39466),
AAO3 (comp43593), and ABA2 (comp714908) were all
identified as up-regulated DETs. Consistent with this
finding, carotenoid cleavage dioxygenase (CCD, comp40565),
which yields another carotenoid-derived phytohormone
from carotenoid and therefore competes with NCED for
the same substrate, was down-regulated, further implicating
enhanced ABA content in the responses of K. caspica to
Although ABA is predominantly biosynthesized and
metabolized in vascular tissues, it acts in the stomatal
Unigenes No. and % indicate the number and the percentage of unigenes in
each pathway from 4023 differentially expressed unigenes mapped to
responses of distant guard cells [
]. In this way, ABA
intercellular regulation and transport are critical for plant
responsiveness to osmotic stress. It was recently reported
that two G-type ATP-binding cassette (ABC) transporter
genes, AtABCG25 and AtABCG40, encode proteins
responsible for ABA transport and response in Arabidopsis
]. The ABC transporter is conserved in many model
species from E. coli to humans and was reported to
transport various metabolites and signaling molecules through
the action of phytohormones in an ATP-dependent
]. Several different types of ABC transporters
(Table 2) were identified as up-regulated DEGs (differentially
expressed genes) in K. caspica through BLAST homology
searches, but only one unigene (comp306780) belonged to
the G-type ABC subfamily. Nevertheless, this finding does
not preclude the possibility that other non-G-type ABC
transporters or non-ABC transporters identified in K.
caspica contribute to cell-to-cell ABA vesicular transport. In
fact, two nitrate transporter 1/peptide transporters (NRT1/
PTR) family members involved in the transport of nitrogen
(N) compounds were recently characterized as novel ABA
transporters in Arabidopsis despite being wholly distinct
from ABC transporter family members .
ABA sensing and signaling
In one proposed model of ABA signaling in Arabidopsis,
PYR/RCAR/PYL (Pyrabactin Resistance/Regulatory
Component of ABA Receptor/Pyrabactin Resistance 1-Like)
family proteins, which act as ABA receptors, recognize
and bind to group A PP2C (type 2C protein phosphatase)
molecules in the presence of ABA. Subclass III SnRK2s
(SNF1-related protein kinase 2) are then released from
PP2C-dependent negative regulation, allowing the
activated SnRK2s to phosphorylate downstream proteins such
as ABA-responsive element (ABRE)-binding transcription
factors (ABFs) [
]. Similar regulation of ABA
signaling has been detected in other species such as wheat
]. In K. caspica, orthologs of PYL (comp365319,
comp38298), PP2C (comp6211), and SnRK2 (comp 30500,
comp27671, comp38192) were up-regulated in the salt
stress sample (Figure 4), indicating that the regulation of
ABA signaling is indeed conserved among plant species.
Based on phylogenetic analysis, the DEGs comp365319,
comp38298, and comp907276 in K. caspica grouped
together with AtPYL4-9 (Figure 4). Since AtPYL4, AtPYL5,
AtPYL6, AtPYL8, and AtPYL9 have been found to inhibit
PP2Cs even in the absence of ABA [
] and considering
that the ectopic expression of PYL5 and PYL8 in
Arabidopsis results in enhanced drought resistance [
comp365319, comp38298, and comp907276 may function
independently of ABA as positive regulators in the salinity
response of K. caspica. Moreover, AtHAI1, AtHAI2, and
AtHAI3 (Highly ABA-Induced PP2Cs) and the DEG
comp6211 in K. caspica made up the clade A PP2Cs
(Figure 4), which had the greatest effect on
ABAindependent low water potential phenotypes and less of
an effect on classical ABA sensitivity phenotypes .
Thus, comp6211 was associated with known clade A
PP2Cs in ABA-independent salinity-associated signaling.
Furthermore, physiological analyses illustrated that the
DEGs comp27671, comp30500, and comp38192 were
classified into groups I, II, and III, respectively (Figure 4). Of these,
only group III SnRKs are considered ABA-dependent
]. It nevertheless remains possible that
many of the K. caspica DEGs may be involved in
ABAindependent salinity-signaling cascades.
Real-time PCR validation of differentially expressed unigenes
To validate the transcriptome data of K. caspica under
salt stress, five DEGs that were found to be up-regulated
in K. caspica exposed to salt stress, namely, orthologs of
SnRK2 (comp38192, comp 30500), PYL (comp38298,
comp365319), and PP2C (comp6211), were selected for
real-time PCR analysis using two-month-old K. caspica
seedlings treated with 300 mM NaCl. As shown in
Figure 5, all five genes exhibited enhanced expression at
certain points during the treatment period, confirming
the comparative transcriptome data for salt-stressed K.
caspica. Comp6211 and comp30500, which are predicted
to be ABA-independent signaling components, also
changed over the course of the treatment period, implicating
their functions in K. caspica responses to salinity. As ABA
receptors, PYL (comp38298, C; comp365319, D)responded
earlier in the presence of salinity stress, recognized and
bind to group A PP2C (comp6211, E) molecules. Subclass
III SnRK2s (comp38192, A; comp 30500, B), which
responded to salinity stress at 12 h, a little later than PYL
(comp38298, C; comp365319, D) and PP2C (comp6211, E),
are then activated, allowing the SnRK2s to phosphorylate
downstream proteins. And the expression of the five genes
was comparable with the fold change estimated by
transcriptomic data illustating that the changes presented in
real-time PCR are biologically significant. Taken together,
these results validated the involvement of ABA in K.
caspica subjected to salt stress. Actually, ABA is universal as
stress-invlolved hormone in plant kimdom. Besides, the core
components in ABA signaling have been obtained in rice,
Selaginella moellendorffi, Physcomitrella patens,
Ostreococcus tauri [
]. Although the PYR/PYL/RCAR–PP2C–
SnRK2 pathway model has been established [
], it is not
clear whether this model can explain all ABA responses in
plant kindom. It is necessary to determine whether these
redundant variants are dependent on or independent of the
core ABA pathway among different plants.
Other DGEs involved in other hormone signaling
Plants adapt to adverse environments by integrating growth
and development to environmentally activate cues. Besides
ABA, several DGE involved in other hormone signaling
indicated multiple hormone crosstalks in K.caspica responses
to salt stress. For example, GA integrates generic responses
into abiotic stress tolerance via the DELLA proteins [
(comp30323, comp47577) to regulate plant development.
Auxin modulate plant development,especially root system
architecture, to defense for salt stress by SAUR family
protein (comp 31574, comp181105, etc.) and auxin responsive
GH3 gene family (comp324952, comp9514, etc.) [
Brassinosteroid (BR) and ethylene, which respectively
mediated by BR-signaling kinase (comp768925), ethyle-reponsive
transcription factor (ERF1, comp20087; ERF2, comp382497),
and EIN3-binding F-box protein (EBF1-2, comp768925), are
also involved in strateges plants take to cope with
]. Therefore, the response to salt stress
in K.caspica is a comprehensive regulatory network in term
of the plant hormones signaling cascade, and need further
This study profiled the transcriptome of K. caspica
under salt stress using Illumina RNA-seq technology to
identify responsive genes and specific pathways involved
in the salinity response of K. caspica. The transcriptome
profile data provide a foundation for further investigation
of the molecular basis underlying salt stress tolerance in
this species. Several key genes involved in ABA
metabolism, transport, sensing, and signaling functions were
found to be induced by salt stress treatment. Thus, the
transcriptome profiling approach and subsequent gene
expression analysis in K. caspica provide important clues for
the identification of functional genes and the contribution
of the ABA signaling pathway to the salt tolerance of
Additional file 1: Table S1. Primer pairs used for real-time.
Additional file 2: Table S2. Overview of de novo assembly statistics for
K. caspica transcriptome sequencing.
Additional file 3: Table S3. Detailed information for 216,415 unigenes
annotated in the K. caspica transcriptome.
Additional file 4: Table S4. Overview of unigene annotation statistics
collected from the NCBI nr, Swiss-Prot, TrEMBL, CDD, pfam, and KOG
Additional file 5: Table S5. Detailed information for 60,127 differentially
expressed unigenes between the control and salt stress samples.
Additional file 6: Table S6. KEGG functional analysis of the differentially
The authors declare that they have no competing interests.
ZX carried out the molecular genetic studies, participated in the design
of the study, performed the data analysis, and drafted the manuscript.
LM carried out the sequence alignment and performed the data
analysis. CD participated in gene expression assay. ZF conceived of
the study, and participated in its design and coordination and helped
to draft the manuscript. All authors read and approved the final
This work was supported by the National Natural Science Foundation of
China (No. 30960035) and the 973 Pre-Research Project of the Ministry of
Science and Technology, China (2012CB722204).
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