Keywords:
Apocyni Veneti Folium; correlate analysis; halophyte; proteomics; salt
tolerance
INTRODUCTION
Soil salinity is a prevalent abiotic stress. High levels of salt can
cause metabolism disorders, osmotic stress, ion imbalance, mineral
nutrient deficiency, water uptake interference, oxidative damage, toxic
ions accumulation and eventually growth inhibition (Pi et al. ,
2018; Yang et al. , 2013). To reduce these detrimental effects
under salt stress, evidence shows that plants have evolved complex
physiological and molecular responses allowing for adaptation, including
selection of ion uptake and exclusion, compartmentalization of
Na+ in vacuoles, synthesis of compatible solutes,
adjustment of photosynthesis, detoxification of reactive oxygen species
(ROS) and regulation of specific protein expressions (Zhang et
al. , 2012). Moreover, halophytes have developed unique structures that
allow them to grow under severe salt stress conditions (Chen et
al. , 2018). An improved understanding of molecular responses to NaCl
treatment may therefore facilitate the development of plants with
increased tolerance to salt stress.
Apocynum venetum L. (AVL), a well-known medicinal halophyte, has
attracted much attention in terms of its antioxidant properties. Modern
pharmacological studies have demonstrated that Apocyni Veneti Folium
(AVF) has several pharmacological functions, such as anti-hypertension,
anti-depressant and hepatoprotection (Xie et al. , 2012). Although
AVL exhibits a high physiological plasticity for salt tolerance, its
growth is affected by salt stress (Chen et al. , 2018; Xieet al. , 2012). Our previous studies have shown that this
medicinal herb can tolerate 300 mM NaCl treatment, under which a total
of 51 metabolites exhibited significant alteration (Chen et al. ,
2019b). Although these salt-responsive metabolites are important, plant
salt tolerance is controlled by sophisticated signaling and metabolic
networks. Therefore, it is necessary to establish a truly meaningful
protocol to effectively and systematically reveal the mechanism of salt
tolerance and quality formation of AVF.
High-throughput transcriptomics of identifying salt-responsive genes and
molecular regulatory pathways has contributed to our understanding of
salinity stress in species including Arabidopsis (Stanley Kim et
al. , 2005), Jerusalem artichoke (Zhang et al. , 2018) and
soybean (Liu et al. , 2019). However, the transcriptome data may
not correlate well with the results from proteomic analysis because mRNA
levels are not always correlated to those of corresponding proteins due
in part to post-transcriptional and post-translational modifications.
Previous evidence showed that only poor or moderate correlation between
the two levels of expression was found in species (Mooney et al. ,
2006). Proteomic analysis is a tool that facilitates the study of global
protein expression and provides a large amount of information about the
individual proteins involved in specific biological responses. Proteomic
profiles have been built for different species, including Arabidopsis
(Jiang et al. , 2007), sugar beet (Wu et al. , 2018),Aeluropus lagopoides (Sobhanian et al. , 2010), soybean (Piet al. , 2016), Salicornia europaea (Wang et al. ,
2009), sesame (Zhang et al. , 2019), Panax ginseng (Kimet al. , 2019) as well as spinach (Li et al. , 2019).
Additionally, isobaric tags for relative and absolute quantification
(iTRAQ) have become a powerful tool in quantitative proteomics,
especially for hydrophobic and low-abundant proteins in cells and
organelles under different environmental conditions (de Abreu et
al. , 2014; Liu et al. , 2014b; Wang et al. , 2013; Wanget al. , 2019), such as, salt-stressed Kandelia candel(Wang et al. , 2013), Jerusalem artichoke (Zhang et
al. , 2018) and Arabidopsis (Pu et al. , 2019), drought-stressed
cassava (Ding et al. , 2019), virus-infected tobacco (Das et
al. , 2019) and heavy-metal-stressed Typha angustifolia (Bahet al. , 2010). However, in spite of the progress underlying
salt-tolerant mechanisms based on physiology and metabolomics (Chenet al. , 2019b; Chen et al. , 2018), a comprehensive
description of the proteome changes and gene transcription is lacking.
In this present study, we combined transcriptomics based on RNA-seq
technology with proteomics based on iTRAQ platform, and then integrated
the results with previous study on
metabolomics and physiology (Chenet al. , 2019b). The generalized workflow was shown in Fig. 1. On
the basis of bioinformatics analysis, 300 differentially expressed
proteins (DEPs) were categorized and analyzed. Then, quantitative
reverse transcriptase-polymerase chain reaction (qRT-PCR) results and
transcriptomics altogether were used to validate the expression levels
between transcripts and their corresponding proteins. Gaining knowledge
of salt tolerance in AVF from integrated transcriptomics, proteomics and
metabolomics provides insights into the molecular basis of plant salt
tolerance, which ultimately leads to quality improvement.
MATERIALS AND
METHODS
Plant material and salt
treatment
Salt stress experiments have been described in detail in our previous
articles (Chen et al. , 2019b; Chen et al. , 2018). Briefly,
the experiment was carried out in the shelter covered by a transparent
film that blocked rainwater, while other conditions were similar to the
open-air environment in Nanjing University of Chinese Medicine. The main
roots of AVL (two years old and originated from the same plant) were
planted in pots filled with 25 kg of soil. The parameters of soil were
as follows: texture, loam; organic carbon, 36.6 g
kg-1; cation exchange capacity, 17.0 cmol(+)
kg−1; pH, 5.0. Salt stress tests were conducted when
AVL was about 30 cm height. Four groups were exposed to different levels
of salt treatment, 0 (control, watering), 100 (low stress), 200
(moderate stress) and 300 (high stress) mM NaCl, respectively. All
groups were designed with 9 replicates and 3 pots per replicate by
pouring 2 L of solution. NaCl concentrations increased gradually by 50
mM every four days to reduce osmotic shock until the designated
concentration was reached and the treatments were lasted for 6 times (20
days). After 12 h of the last salt treatments, leaves were harvested.
After being quick frozen in liquid nitrogen, these leaves were
transferred to a -80 °C refrigerator for storage.
Transcriptome analysis
Total RNA was extracted from 100 mg leaves according to the
manufacture’s protocol using RNAprep Qubit RNA kit and Agilent
Technologies 2100 Bioanalyzer. The RNA integrity and quality was
confirmed by a NanoDrop ND-1000® spectrophotometer (GE
Healthcare™) and electrophoresed in 1 % agarose gel. Then, poly(A) mRNA
was purified with Oligod(dT) beads and fragmented. After synthetization
of first-strand cDNA using reverse transcriptase and random primers,
second-strand cDNA was synthesized using DNA polymerase Ⅰ. Four groups
were subjected to end repair and addition of a single A base and
ligation with adapters. Suitable fragments were amplified through PCR to
create a library for sequencing using Illumina HiSeqTM2500. No reference genomes were used for AVF transcriptomic analysis.
Clean reads were obtained by ngsQCToolkit-2.3.32 for purification and
filtration of low-quality sequences of the raw data. High-quality reads
were assembled with Trinity software to construct a unigene library
after further filtration. The unigenes showed an average ratio-fold
change |log2FC|≥1 and FDR≤0.05 were
confidently considered as differentially expression genes (DEGs). The
raw data of transcriptomes converting to proteome library
Protein extraction, digestion, iTRAQ labeling and strong
cation
exchange
To ensure the ability to conduct statistical analyses, two biological
replicates (12 plants per replicate) were used for the iTRAQ-based
quantitative proteomic analysis. The procedure for protein extraction
was modified from the phenol method (Li et al. , 2015; Wanget al. , 2013). In short, samples of AVF (about 100 mg) were
vortexed with 10 mL extraction buffer. The extraction was performed
under shaking, followed by centrifugation. The phenol phase transferred
was re-extracted under the same conditions; this step was repeated once
more. After precipitation and resuspension, proteins were rinsed, and
then transferred into a new tube and centrifuged. Protein pellets were
dissolved in RIPA buffer and centrifuged. Finally, the pellets were
air-dried and ready for use. The protein concentrations were determined
using the BCA method.
Proteins were digested according to the FASP method (Hua et al. ,
2016; Lan et al. , 2011; Wang et al. , 2013). iTRAQ labeling
was performed according to the manufacturer’s instructions (Applied
Biosystems). The control samples’ replicates were labeled with tags 113
and117, and the salt-treated labeled with tags 114 and 118 (100 mM salt
treatment), tags 115 and 119 (200 mM salt treatment), tags 116 and 121
(300 mM salt treatment), respectively. After labeling, individual iTRAQ
8-plex samples were mixed and diluted into 0.1% trifluoricacetic acid,
followed by loading on a C18 reverse phase mini-column.
After washing and elution, the eluates were dried down and dissolved by
strong cation exchange (SCX) solvent A (25% v/v acetonitrile, 10 mM
ammonium formate, pH 2.8). The peptides were eluted by 20 mM
HCOONH4 (pH 10) and 20 mM HCOONH4 and
80% acetonitrile (pH 10) for phase B, and UV wavelength was set at 214
nm and 280 nm with a flow rate at 0.2 mL min-1.
Tandem mass spectrometry
analysis
The fractionated samples were lyophilized to remove acetonitrile and
resuspended in 5% acetonitrile and 0.1% formic acid, and then
centrifuged. The nano LC–MS/MS was carried out using a Thermo
Scientific Q Exactive. The peptide mixture was loaded on an Acclaim
PepMap RSLC C18 (75 μm × 150 mm, 2 μm, 100 Å). 0.1%
formic acid was used as buffer A and 80% acetonitrile/0.1% formic acid
as buffer B. The peptides were eluted at a flow rate of 0.3 mL
min-1 with a gradient (B%): 0–4% for 5 min,
4%–50% from 5 to 45 min, 50%–90% from 45 to 50 min, and 4% for 15
min.
MS data analysis and protein
identification
MS data were performed using Proteome Discoverer 1.3 software. To
increase the confidence level, proteins with an iTRAQ ratio higher than
20 or less than 0.05, or the absolute value of coefficient of variation
(C.V.) larger than 0.5 were not considered as quantified (Zhang et
al. , 2019). DEPs were selected based on the following criteria:
proteins in which the mean ratio corresponding to the protein reporter
ion intensity originating from salt-treated protein samples with respect
to fully control protein samples had an average ratio-fold change ≥1.5
or ≤0.67, respectively; p ≤ 0.05 (Zhao et al. , 2016).
Bioinformatics analysis
Open reading frame of AVF obtained from the sequences of the
transcriptome was established into a protein database. Then, the
sequences of protein database was compared with the databases of
Uniprot, Swissprot,
NCBI,
NR, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG)
and Clusters of Orthologous Groups of proteins (COG). GO analysis of
proteins was carried out using QuickGO software which was performed
using BLASTp search against Uniprot to find the ID of proteins and then
annotate them, generating molecular function, biological process, and
cellular component information. KEGG database was used to take advantage
of the current knowledge of biochemical pathways and other types of
molecular interactions (Hua et al. , 2016). DEPs were classified
according to GO, KEGG and STRING to predict functions, significant
pathways and protein-protein interaction.
Total RNA extraction and qRT-PCR
analysis
To examine relationships between mRNA expression and protein abundance
under salt stress, the relative transcript level of nine salt-responsive
proteins was examined by qRT-PCR. Briefly, total RNA was extracted from
salt-treated and control AVF by plant RNA kit (R6827-01, Omega Bio-Tek),
and cDNA was reverse transcribed from 1 μg of total RNA using a first
strand cDNA synthesis kit (Yeasen). Gene-specific primers used for
qRT-PCR were designed using Primer 5 software according to cDNA
sequences obtained from the AVF RNA-seq (Table S1). The truncated
beta-actin gene was used as an endogenous control for normalization. The
PCR reaction was carried out in a 20 μL volume containing 10 μL 2
×Hieff® qPCR SYBR Green Master Mix reagent (Yeasen), 2
μL template cDNA, 0.4 μL of each primers and 7.2 μL sterilized water
with the following reaction conditions: 95 °C for 5 min; followed by 40
cycles of 95 °C for 10 s; 60 °C for 30 s. Three biological replicates
(12 plants per replicate) were used. Relative gene expression was
calculated using the formula 2−∆∆Ct (Li et al. ,
2015).
Correlation between DEGs and
DEPs
The correlation between transcripts and protein abundance in each salt
treatment compared to control was achieved by SPSS 14.0 software.
Data analysis
Statistical analyses of data among groups were performed using Student’st -test at a 0.05 level. Statistical analysis was performed by
one-way analysis of variance. Data are presented as means ±SD of three
replicates.
RESULTS
iTRAQ primary data analysis and protein
detection
Based on the iTRAQ analysis, a total of 28775 spectra were generated
from the iTRAQ experiment. By matching to known spectra, 8087 unique
peptides and 4344 proteins were detected. Summary of the iTRAQ
information was shown in Fig. 2. There were 13977, 24024, 3478, and 509
peptides with 4–10, 11–20, 21–30, and more than 30 peptide length,
respectively (Fig. 2A). Isoelectric point of the majority identified
protein was between 4.5and 10 (Fig. 2B), and over 84.9% of the proteins
included at least two peptides (Fig. 2C). Mass distribution of the
identified protein species showed that more than half of them were 20-80
kDa (Fig. 2D). In addition, sequence coverage of the majority of
identified protein species was less than 30% (Fig. 2E).
Identification and functional classification of
DEPs
Based on the criteria mentioned in the experimental section, 143, 162
and 167 DEPs were found in comparison between salt-stressed samples and
control (Fig. 3A), of which 40 were shared in three comparisons. In
detail, there were 85, 110 and 99 up-regulated and 58, 52 and 68
down-regulated proteins for low, moderate and high level of salt
treatments versus control, respectively (Fig. 3B).
All salt-responsive DEPs in different levels of salt stress were
classified by GO annotation software into three functional groups:
molecular function, biological process and cellular component (Fig. 3C).
On the basis of biological process analysis, most of the DEPs were found
to be related to small molecule metabolic process, biosynthetic process,
response to stress, carbohydrate metabolic process, translation and
catabolic process. In addition, most of the annotated molecular
functions were found to be related to ion binding, oxidoreductase
activity, structural constituent of ribosome and hydrolase activity. In
the category of cellular component, thylakoid, intracellular, plastid
and ribosome were the most represented.
KEGG and STRING analysis of
DEPs
The KEGG analysis demonstrated that metabolic pathways, biosynthesis of
secondary metabolites, photosynthesis, biosynthesis of antibiotics,
ribosome were prominently affected. A total of 300 proteins participated
in 152 pathways. E.g., 32, 44 and 42 DEPs were involved in metabolic
pathways for the low, moderate and high level of salt stress,
respectively; 18, 27 and 33 DEPs were involved in biosynthesis of
secondary metabolites, respectively, followed by 9, 7 and 5 DEPs related
to photosynthesis, respectively (Fig. 4). Obviously, DEPs were involved
in more pathways with the increasing levels of salt treatments.
The function of proteins is vital to cells. To predict the protein
interactions, functional relations and networks among DEPs using STRING
software (Fig. 5). It can be observed that extensive interactions were
found amongst ribosomal proteins (RPs) and heat shock proteins (HSPs),
or between HSPs and other proteins with the enhanced levels of salt
stress. These results indicate that RPs and HSPs probably play roles in
protecting protein functions under salt stress conditions.
Shared DEPs of AVF in response to salt
stress
The relative abundance and functional properties of a list of 40 DEPs
shared in three comparisons was given in Fig. 6A. The main molecular
functions were ion binding, followed by oxidoreductase activity (Fig.
6B), and the main biological processes were related to response to
stress, followed by carbohydrate metabolic process (Fig. 6C). KEGG
pathways showed that photosynthesis, ribosome, nitrogen metabolism,
biosynthesis of secondary metabolites, carbon metabolism and
phenylpropanoid biosynthesis, were altered under salt tolerance (Fig.
6D). In addition, the STRING analysis revealed that shared DEPs, such as
HSPs and RPs were interacted in response to salinity (Fig. 6E). Taken
together, shared DEPs in three comparisons probably play important roles
role in improving AVF salt tolerance.
Transcriptional analysis by
qRT-PCR
Nine DEPs, selected from several enriched pathways, such as peroxisome,
phenylpropanoid biosynthesis, photosynthesis, ribosome, Foxo signaling
pathway and plant-pathogen interaction, were examined between mRNA
expression level and protein abundance by qRT-PCR
(Fig.
7). Results showed the expressions levels of six genes,
Tr_AVENL_20218 (uncharacterized protein), Tr_AVENL_23089 (ATP
synthase subunit delta, chloroplastic), Tr_AVENL_3558 (ruBisCO large
subunit-binding protein subunit beta, CPN60B), Tr_AVENL_4882 (catalase
isozyme 1-like, CAT), Tr_AVENL_23055 (pathogenesis related protein,
PRH) and Tr_AVENL_972 (dehydrin 1) were consistent with the
corresponding DEPs abundance, indicating that these proteins were
regulated at the transcriptional level. While, the transcript levels of
the remaining three genes (Tr_AVENL_17965, Tr_AVENL_25878 and
Tr_AVENL_27306) were inconsistent with the protein expression levels.
In addition, the result of qRT-PCR was consistent with that of
transcriptome with the exception of Tr_AVENL_20218 and
Tr_AVENL_23089 (Fig. 8). Specifically, the qRT-PCR result of
Tr_AVENL_20218 was up-regulated while it was down-regulated in
transcriptome under low level of salt stress. Similarly, up-regulated
level of Tr_AVENL_23089 in qRT-PCR result under moderate and high
levels of salt stress was found to be inconsistent with the
transcriptome analysis.
Correlation analysis between transcripts and
proteins
We generated RNA‐seq data from the same samples. After further
identification DEGs under salt treatments by comparing with control, a
total of 807, 1512 and 1258 DEGs were obtained in three comparisons,
respectively. 23, 16 and 11 DEGs/DEPs showed significant correlation in
gene expression and protein levels under low, moderate and high levels
of salt stress compared to the control, respectively, showing increasing
correlations (0.29, 0.57 and 0.63, respectively) (Fig. 9A-C). Fig. 9D
revealed a weak correlation (R = 0.49) in 49 DEGs/DEPs found in the
salt-stressed AVF in comparison with control , and four DEPs shared in
three comparisons, including dehydrin 1, annexin, PRH and prolyl
oligopeptidase (POP) (Fig. 6A), were contained in these significant
correlated DEGs/DEPs. Therefore, these DEPs may play active roles role
in AVF tolerance to salt stress.
DISCUSSION
Halophytes can complete their life cycle at salinities above 200 mM NaCl
(Flowers, 2004). Meanwhile, molecular mechanisms of halophytes in
response to salt stress revealed that some plants evolved specific
salt-tolerant mechanisms (Wang et al. , 2009). Our previous study
on metabolomics analysis (Chen et al. , 2019b) indicated that AVF
exposure to low level of salt stress had the strongest ability to adapt
to salt shock, followed by the moderate level one, and the summarized
data was shown in Fig. S1. In this study, combined with transcriptomics,
proteomics and metabolomics, we focused our analysis on the DEPs related
to carbohydrate and energy metabolism, lipid and amino acid metabolism,
biosynthesis of secondary metabolites, signal transduction,
transcription, translation, as well as protein folding and degradation
and provide insight into AVF salt tolerance (Table S2).
Proteins related to carbohydrate
metabolism
In many plants, proteins associated with carbohydrate metabolism altered
expression patterns severely under salt stress (Yang et al. ,
2013; Zhang et al. , 2012). In our study, a large proportion of
DEPs involved in carbohydrate metabolism, such as
glycolysis/gluconeogenesis, citrate cycle, starch and sucrose
metabolism, pentose phosphate pathway and carbon fixation in
photosynthetic organisms, was altered the abundance; many of them were
up-regulated, such as beta-glucosidase 44-like (BGLU),
fructose-bisphosphate aldolases (FBAs), triosephosphate isomerase (TPI),
two malate dehydrogenases (MDH), phosphoglycerate kinase (PGK) and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PK),
beta-galactosidase 1 (BGAL1), alpha-amylase (AMY), two chitinase family
proteins,
serine-glyoxylate
aminotransferase (sgaA), glucose-1-phosphate adenylyltransferase (APS)
and UN (gi|661880779). Specifically, BGLU is a key enzyme in
the cellulose hydrolysis process causing abscisic acid-glucose conjugate
hydrolysis (Yang et al. , 2013). The activity of an extracellular
BGLU was up-regulated under moderate level of salt stress, which was
consistent with the reports that it was highly induced in barley (Dietzet al. , 2000) under salt stress. Furthermore, the overexpression
of BGLU genes regulated endogenous abscisic acid (ABA) levels during the
development of watermelons under drought stress (Li et al. ,
2012). Similarly, the relative abundance of two FBAs was accumulated
under high level of salt stress. Similar results were found in
heat-stressed alfalfa seeds (Li et al. , 2013) and grape leaves
(Liu et al. , 2014b) and NaCl-stressed sugar beet (Yang et
al. , 2013). Chloroplastic TPI showed significant accumulation in
response to moderate and high level of NaCl, consistent with sugar beet
under 200 mM salt stress (Yang et al. , 2013). In particular, MDH
reversibly catalyzed the oxidation of malate to oxaloacetate and the
overexpression of MDH gene enhanced the synthesis of organic acids and
conferred tolerance to aluminum in transgenic alfalfa (Tesfaye et
al. , 2001). In this study, the expression of MDH was increased under
low and moderate levels of salt stress, consistent with the result of
salt-treated sugar beet roots (Yang et al. , 2013) and
drought-treated maize (Wang et al. , 2019). However, this enzyme
was decreased in sugar beet (Wu et al. , 2018) and upland cotton
(Li et al. , 2015) under salt stress conditions. A possible reason
was that the MDH expression was diverse in each species under various
stresses. A Calvin cycle related protein, GAPDH showed an increase in
response to moderate level of NaCl. Two chloroplastic GAPDHs were
accumulated in salt-treated sugar beet (Yang et al. , 2013) and
virus-infected Nicotiana tabacum plants (Das et al. ,
2019), but were depressed in drought-treated cassava (Ding et
al. , 2019) and salt-treated rice as well (Chitteti and Peng, 2007). PK
showed increased in abundance under moderate level of salt stress, but
remained unchanged under low and high ones. The results were similar
between two contrasting salinity-tolerance sesame genotypes in response
to salt stress (Zhang et al. , 2019). Taken together, the
increases of glycolysis and TCA cycle related enzymes implied the
enhancement of respiration under salt stress.
In many model plants, overall carbon metabolism and the levels of
sucrose and starch were severely affected under salt stress (Wanget al. , 2013; Zhang et al. , 2012), which possibly results
from a metabolic shift toward sucrose production. High salinity also
altered the substantial accumulation of starch grains in chloroplasts.
Several proteins in this study were related to starch metabolism, such
as sucrose synthase (SUS), ADP glucose pyrophosphorylase (AGP), BGLU,
AMY as well as an UN (gi|661880779). We found here that AMY, an
enzyme involved in the degradation of starch to sucrose, was increased,
which was agreed with the metabolomics profiles that the content of
sucrose was increased under severe salt stress and this would favor salt
tolerance. Consistent with our result, this enzyme was induced inPanax ginseng leaves upon exposure to heat stress (Kim et
al. , 2019). Particularly, an up-regulated UN (gi|661880779) is
known as exhibiting hydrolase activity and also plays a role in starch
and sucrose metabolism and phenylpropanoid biosynthesis (Denoeudet al. , 2014). Our aforementioned results demonstrated that the
accumulated chloroplasts and soluble sugar were possibly employed to
overcome salinity stress. After salt stress, the accumulated starch can
be transformed into sucrose, which might be a consequence of a
significantly up-regulated expression of the gene encoding. Furthermore,
it is worth noting that the most altered protein was the up-regulated
chitinase with 8.6- and 9.4-fold change under moderate and high level of
salt treatments, respectively. Reports showed that the up-regulated
chitinase improved salt tolerance in Thaliana by preventing the
excessive accumulation of Na+ (Kwon et al. ,
2007).
However, several proteins were found to be down-regulated, such as
peroxisomal (S)-2-hydroxy-acid oxidase GLO4-like isoform X5 (GLO4) under
low level of salt stress, galactinol synthase 1 (GOLS1) under moderate
level of salt stress and four proteins, probable rhamnose biosynthetic
enzyme 1, SUS, phosphoenolpyruvate carboxykinase [ATP]-like (PCK)
and an UN (gi|661898708) under high level of salt stress.
Specially, GLO4 is involved in glyoxylate and dicarboxylate metabolism
under salt stress (Zhang et al. , 2019). GOLS1, a stress-related
protein, plays a key regulatory role in the carbon partitioning;
overexpression of an Arabidopsis GOLS family member caused an increase
in the levels of endogenous galactinol and raffinose (Taji et
al. , 2002). In addition, the up-regulated GOLS could be found in
cold-stressed Phaseolus vulgaris seeds (Liu et al. , 1998)
and drought-stressed cassava (Ding et al. , 2019). In agreement
with our research, PCK was increased in abundance in two sesame
genotypes after salinity tolerance (Zhang et al. , 2019).
Obviously, with the salt concentrations increase, the affected proteins
increase to survive and adapt, supported by metabolomics results of
quite high photosynthesis activity under salt stress (Chen et
al. , 2019b).
Proteins related to energy
metabolism
DEPs of ferredoxin–NADP reductase (PETH), two FBAs, alanine:
glyoxylate aminotransferase isoform 1, two carbonic anhydrases (CAs),
glutamine synthetase (GLN) and ferredoxin– nitrite reductase (NIR),
related to energy metabolism were increased in abundance with the
exception of glutamate dehydrogenase (GDH) and cysteine synthase (OASA).
These DEPs were also involved in photosynthesis, carbon and nitrogen
metabolisms and pentose phosphate pathway. Many studies have found that
nitrogen metabolism plays an important role in the complex process of
plant response to salt stress (Chen et al. , 2019c). Specifically,
GLN functions as the major assimilatory enzyme for ammonia, and GDH
works as a link between carbon and nitrogen metabolism. Therefore, the
up-regulated GLN and down-regulated GDH can be a way to prevent
excessive accumulation of ammonium in AVF cells, and high level of salt
stress probably disturbed nitrogen transformation considering the
enhanced glutamine in proteomics data (Wu et al. , 2018). In
addition, NIR and two CAs involved in nitrogen metabolism were greatly
enhanced in response to NaCl, suggesting an increase in the assimilation
of nitrogen through nitrate reduction; inconsistently, the expression of
NIR homologues in salt-stressed soybean seedlings (Liu et al. ,
2019) and drought-stressed cassava was severely inhibited (Ding et
al. , 2019).
Photosynthesis is generally considered to be salt sensitive. PETH showed
significant accumulation in response to severe salt stress, implying the
adjustment of ATP synthesis, and similar result was taken place in the
sugar beet (Yang et al. , 2013). Reports on some Calvin cycle
related proteins, such as
CAs,
were increased in Salicornia europaea (Wang et al. , 2009)
but were decreased in halophyte Aeluropus lagopoides (Sobhanianet al. , 2010) under salinity. Different species may utilize
varying light reaction strategies to cope with salinity (Yu et
al. , 2011); however, maintaining an energy supply is indispensable for
plants to reduce salt stress injury. It is further supported by our
proteomic results in which the levels of proteins carrying out
light-dependent reactions were increased with higher salinity.
Proteins related to lipid and amino
acid
metabolism
Metabolic adjustments play important roles in attaining a new balance of
energy and metabolites. We found that a large number of DEPs was
enriched in fatty acid metabolism, linolenic acid metabolism and
arachidonic acid metabolism. Four members of lipoxygenases were
increased in abundance, and studies showed that the increased level of
lipoxygenase 2 contributes to a protective effect against increased
salinity in rice (Liu et al. , 2014a). Antioxidant system in lipid
metabolism, including some members of ascorbate peroxidases (APXs),
glutathione peroxidases (GPXs) and glutathione S-transferases (GSTs)
responded to various stresses (Jiang et al. , 2007) was altered.
Several GPX genes in Arabidopsis were up-regulated coordinately in
response to stress (Rodriguez Milla et al. , 2003), but were
down-regulated in cassava (Ding et al. , 2019). The flexibility
and enhancement of lipid metabolism helps AVL survive under severe
condition.
The iTRAQ data showed that 31 DEPs were related to amino acid
metabolism; most of them increased in abundance under low and moderate
level of salt stress. Pyrroline-5-carboxylate synthetase (P5CS) is
involved in the proline synthesis to overcome salt stress, and the
enhanced abundance of this protein is consistent with proline content
(Chen et al. , 2018). The up-regulated level of methionine
synthase (MET) and ASP were supported by salt-stressed sugar beet (Yanget al. , 2013) and sesame (Zhang et al. , 2019),
respectively.
However, several DEPs were notably inhibited under severe stress, such
as 4-coumarate: CoA ligase 3 (4CL3), which can be used to synthesize
several phenylpropanoid-derived compounds. Remarkably, three
proteins,
phenylalanine ammonia lyase (PAL), cytochrome P450 CYP73A120 (CYP) and
phospho-2-dehydro-3-deoxyheptonate aldolase (aroF), were found to play
important roles in alleviating the damage to plants and decreased in
abundance under severe salt stress. Specifically, PAL is a key enzyme of
plant metabolism based on the phenylpropane skeleton, and CYP family
proteins play critical roles in flavonoid and sterol synthesis. The
expression level of them was significantly suppressed under drought and
salinity tolerance, respectively (Ding et al. , 2019; Yan et
al. , 2014).
In addition, some DEPs related to redox system in amino acid metabolism
were changed, such as CATs, APX2 and GSTs. CATs were decreased in
abundance after salt stress, consistent with the physiological analysis
(Chen et al. , 2018). Many proteomic studies have confirmed the
alteration of this redox related protein (Chen et al. , 2019c).
Known to play a crucial role in glutathione-ascorbate cycle, APX was
increased in AVF under salt stress. However, the abundance of GST was
regulated differently, consistent with GST6 expression in stressed maize
(Zhao et al. , 2016). Therefore, different protein productions
were adopted to alleviate damage to plants.
Proteins related to biosynthesis of
secondary
metabolites
A total of 23 DEPs were related to secondary metabolism. Specially, in
the widely affected phenylpropanoid biosynthesis pathway, the abundance
of nine proteins, including caffeic acid 3-O -methyltransferase
(COMT), 4CL3, peroxidase (POD), PAL, BGLU, CYP, two cinnamyl alcohol
dehydrogenases (CADs) and an UN (gi|661880779) were affected.
The abundance of some proteins was exclusively suppressed under moderate
or high level of salt stress, such as violaxanthin de-epoxidase, UN
(gi|661892013), CAD, chalcone synthase (CHS),
leucoanthocyanidin reductase 1 (LAR1), anthocyanin synthase (ANS),
flavonol synthase (FLS) and UN (gi|661877099). In addition,
four categories of enzymes, including CHS, chalcone isomerase (CHI), CPM
and FLS, play critical roles in flavonoid synthesis under salt stress
(Pi et al. , 2016). Besides, salt tolerance of Arabidopsis and
soybean were positively regulated by CHS and negatively regulated by CHI
and CPM (Pi et al. , 2018). In addition, alkaloid and terpenoid
biosynthesis related proteins were also altered by salt. These
observations were supported by the metabolomics results that more
metabolic alterations were observed as the increasing levels of salt
stress. Some shikimate-phenylpropanoid pathway compounds, such as
flavonoids, phenolic acids and other secondary metabolites were
exclusively decreased by severe salt. In short, high level of salt
stress altered more DEPs related to secondary metabolite biosynthesis
which probably played a crucial role in AVF response to harsh
conditions.
Proteins related to signal
transduction
Multiple signal transduction pathways activate other regulators, and
initiate protective mechanisms through the induction or repression of
functional genes to cope with salt stress (Jiang et al. , 2007).
Most of the up-regulated proteins, such as GLN, HSPs, lactoylglutathione
lyase (GLXI), GAPDH, PRH and some UNs were predominately enriched in the
signal transduction pathways. In particular, the presence of CATs as
well as GLXI indicates fine tuning and efficient decomposition of toxic
byproducts of cellular metabolism (Wang et al. , 2010).
Interestingly, PRH was suppressed under low level of salt stress but
induced under moderate and high ones. Consistent with us, members of
this family protein were suppressed by phytohormones and stress stimuli
(Li et al. , 2015; Wang et al. , 2010), and regulated
differently in salt-treated maize (Chen et al. , 2019c). Moreover,
HSPs play a role in membrane stability using ROS as a signal molecule
(Wang et al. , 2004), and the differently regulated HSPs were
found in flax under heavy metal stress (Kosova et al. , 2011).
Proteins involved in transcription
and
translation
Transcriptional regulation of salt-responsive genes is a crucial part of
the plant response to stress (Jiang et al. , 2007). RNA processing
and ribonucleoprotein complex assembly may possibly represented critical
processes (Lan et al. , 2011). In the present study, the
expression level of small nuclear ribonucleoprotein associated protein B
(SNRPB) was increased in low salt-treated samples, and eight RPs were
increased under at least one level of salt stress. Ribosomes were showed
to be essential ribonucleoprotein complexes engaged in translation
(Zhang et al. , 2018). Observed from the proteomics results of
salt-treated Arabidopsis (Jiang et al. , 2007) and cotton (Liet al. , 2015), the expression levels of some specific RPs
increased while some decreased, suggesting that NaCl stress enhanced
specific protein synthesis if these proteins were of particular
importance to salt tolerance. In addition, eukaryotic translation
initiations (eIFs) involved in protein translation (Jiang and Clouse,
2001), was observed down-regulated under low and moderate level of salt
stress; similar results were found in salt-stressed Arabidopsis (Jianget al. , 2007).
In addition, spliceosome proteins were down-regulated under moderate and
high levels of salt stress. Published articles showed that the
production of ROS and H2O2 triggered
HSP70 synthesis and furtherly enhanced antioxidant enzyme activities
(Tripathy and Oelmuller, 2012). In agreement with us, HSP70s family
members regulated differently in salt-treated sugar beet (Yang et
al. , 2013) and heat-stressed spinach (Li et al. , 2019).
Proteins related to folding and
degradation
Proteins involved in folding and degradation play important roles in
surviving from severe salt treatment. Misfolded proteins bind to
chaperone BiP and are degraded through the proteasome in the disturbed
homeostasis endoplasmic reticulum (Zhao et al. , 2016). DEPs,
Glycosyltransferase (GT), dolichyl-diphosphooligosaccharide (OST48),
calreticulins (CRTs) and two UNs, which promoted the proper folding of
proteins and prevent the aggregation of damaged proteins, were all
ultimately decreased in abundance in at least one NaCl-treated sample.
In particular, CPN60B and CPN60beta2 were increased in abundance in all
salt-treated samples, indicating the activation of chaperones promoted
by stress exposure. Notably, HSPs are often involved in assisting the
folding of de novo synthesized polypeptides, the
import/translocation of precursor proteins, preventing protein
aggregation, maintaining protein functional conformation and cellular
anti-stress ability (Wang et al. , 2004). In addition, members of
other HSPs in AVF were all up-regulated in moderate levels of salt
stress but remains unchanged under low one, suggesting the importance of
molecular chaperones by maintaining proper protein folding and refolding
for salt-tolerance.
Correlation of protein abundance
and gene
expression
A number of transcriptomic studies to explore genome-wide gene
expression reprogramming have been done on salt stress with different
species (Liu et al. , 2019). Under high level of salt stress, the
transcript abundance was more directly relevant to the protein level.
However, an overall weak correlation was in agreement with the general
observation that mRNA levels do not always correlate with protein
levels.
It is worth noting that in three comparisons, four DEGs/DEPs (dehydrin
1, annexin, PRH and POP) were shared in significant correlations,
suggesting their crucial role under salt stress. Specifically, dehydrins
are osmotically active proteins and related to stimulus response. The
accumulation of dehydrin may help compensate increased
Na+ levels in AVF, supported by the salt-treatedHordeum vulgare (Marsalova et al., 2016). Annexin
participation in diverse cellular functions highlight their essential
roles in enhancing multiple stress tolerance (Yadav et al.,
2018). Similar to us, overexpression of annexin genes enhance tolerance
in tomato (Ijaz et al., 2017) and Arabidopsis (Kreps et
al., 2002). As mentioned above, PRH is involved in plant defense
responses to several pathogens and abiotic stresses. In addition, POP is
a proline-specific serine protease, and plays important roles in
multiple biological processes, such as protein secretion, maturation and
degradation of peptide hormones and signal transduction (Tan et
al.,
2013).
However, few reports on salt stress were found. In summary, these four
proteins can be considered as targets and worth of in-depth study for
improving quality engineering of this halophyte.
Molecular mechanisms revealing salt
tolerance in
AVF
On the basis of the identified salt-responsive proteins combined with
physiology and metabolomics, we have revealed the following processes in
AVF surviving from salt stress (Fig. 10). First, enhancement of
photosynthesis and energy metabolism. Second, up-regulation of
antioxidant enzymes. Third, accumulation of osmotic adjustments. Fourth,
enhancement of secondary metabolism. At last, up-regulation of protein
translation and folding. The concerted changes altogether in the above
processes may provide AVF functional advantage under salt tolerance.
We realized that AVF under low level of salt stress could maintain
stronger osmotic regulation ability, synergistic effects of antioxidant
enzymes, energy supply capacity, signal transduction, ammonia
detoxification ability as well as metabolite synthesis. The related
proteins coordinate in energy metabolism and secondary metabolite
biosynthesis in AVF. Furthermore, moderate and high levels of salt
altered more proteins; especially the down-regulated ones related to
biosynthesis of secondary metabolite. Observations also explained why
the quality of low level of salt-stressed AVF was better than other
samples (Chen et al. , 2019a; Chen et al. , 2019b).
CONCLUSIONS
Overall, RNA-seq based transcriptomics and the iTRAQ based proteomics of
AVF were investigated under saline conditions. The purpose was to
identify some salt stress responsive proteins and pathway in AVF and
provided insights into the molecular mechanisms of salinity tolerance
and quality formation. Results showed that the main functions of DEPs
were small molecule metabolic process, biosynthetic process, response to
stress as well as carbohydrate metabolic process. Furthermore, the
diverse array of proteins combined with metabolomics results indicated
that there was a remarkable flexibility in AVF metabolism, such as
enhancement photosynthetic functions and carbohydrate metabolism under
low and moderate level of salt stress and depression biosynthesis of
secondary metabolites under severe stress. In addition, a weak
correlation between the abundance of proteins and the corresponding
transcripts demonstrated that the expression of some proteins could be
regulated by post-transcriptional modifications. In general, the
functional characterization of the proteins/genes revealed by integrated
transcriptomics, proteomics and metabolomics will be helpful for
improved understanding of the molecular mechanisms/networks in AVF and
discovering new targets, and ultimately rationale engineering of
halophytes with enhanced stress tolerance.
ACKNOWLEDGEMENTS
This work was supported by Priority Academic Program Development of
Jiangsu Higher Education Institutions of China (NO.ysxk-2014) and
Postgraduate
Research & Practice Innovation Program of Jiangsu Province
(KYCX18_1606) for the financial supporting.
CONFLICT OF
INTEREST
Authors declare no conflict of interest.
AUTHOR
CONTRIBUTIONS
C.C., W.C., and X.L. designed the experiments and managed the projects.
C.C., Z.L., and Z.C. performed the experiments. Y.H., Y.M., and W.L.
performed the data analysis. C.C. and X.L. wrote the manuscript.
SUPPORTING
INFORMATION
Additional Supporting Information may be found online in the supporting
information for this article.
Figure S1. Venn diagram analysis (A) and hierarchical
clustering analysis (B) of differentially expressed metabolites of AVF
exposed to salt stress compared with control.
Table S1 Specific primer pairs for qRT-PCR analysis
Table S2 Detailed information of key salt responsive DEPs
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