Abstract
It is well known that exogenous trehalose can improve resistances of
plants to some abiotic and biotic stresses. Nonetheless, information
respecting the molecular responses of tobacco leaves to Tre treatment
is limited. Here we show that exogenous Tre can rapidly reduce stomatal
aperture, up-regulate NADPH oxidase genes and increase
O[2]^•-andH[2]O[2] on tobacco leaves at 2 h after treatment. We further
demonstrated that imidazole and DPI, inhibitors of NADPH oxidase, can
promote recovery of stomatal aperture of tobacco leaves upon trehalose
treatment. Exogenous trehalose increased tobacco leaf resistance to
tobacco mosaic disease significantly in a concentration-dependent way.
To elucidate the molecular mechanisms in response to exogenous
trehalose, the transcriptomic responses of tobacco leaves with 10 (low
concentration) or 50 (high concentration) mM of trehalose treatment at
2 or 24h were investigated through RNA-seq approach. In total, 1288
differentially expressed genes (DEGs) were found with different
conditions of trehalose treatments relative to control. Among them,
1075 (83.5%) were triggered by low concentration of trehalose (10mM),
indicating that low concentration of Tre is a better elicitor.
Functional annotations with KEGG pathway analysis revealed that the
DEGs are involved in metabolic pathway, biosynthesis of secondary
metabolites, plant hormone signal transduction, plant-pathogen
interaction, protein processing in ER, flavonoid synthesis and
circadian rhythm and so on. The protein-protein interaction networks
generated from the core DEGs regulated by all conditions strikingly
revealed that eight proteins, including ClpB1, HSP70, DnaJB1-like
protein, universal stress protein (USP) A-like protein, two FTSH6
proteins, GolS1-like protein and chloroplastics HSP, play a core role
in responses to exogenous trehalose in tobacco leaves. Our data suggest
that trehalose triggers a signal transduction pathway which involves
calcium and ROS-mediated signalings. These core components could lead
to partial resistance or tolerance to abiotic and biotic stresses.
Moreover, 19 DEGs were chosen for analysis of quantitative real-time
polymerase chain reaction (qRT-PCR). The qRT-PCR for the 19 candidate
genes coincided with the DEGs identified via the RNA-seq analysis,
sustaining the reliability of our RNA-seq data.
Introduction
Trehalose (Tre), as a non-reducing disaccharide, is formed by two
α-glucose units linked through α, α-1,1-glucosidic bond
(α-D-glucopyranosyl-[[36]1,[37]1]-α-D-glucopyranoside). Tre
biosynthesis and signaling in vivo have been investigated extensively
in many different organisms, including bacteria, yeast, fungi, insects,
plants and animals[[38]1, [39]2]. Even though alternative pathways
exist in different organisms, biosynthesis of Tre typically includes
two steps. Trehalose-6-phosphate synthase (TPS) first catalyses the
formation of trehalose-6-phosphate (T6P) from UDP-glucose and
glucose-6-P, and trehalose-6-phosphate phosphatase (TPP) further
convert T6P into Tre[[40]3]. In vivo, Tre has been reported to protect
the integrity of organelles and cells in some organisms against
enviromental stresses[[41]4–[42]6]. T6P, as an intermediate metabolite
of Tre biosynthesis, has been proved to function as a sensor for in
vivo available sucrose, by this means regulating the responses of
organism to the diverse environmental changes directly, which is
reasonable as the components of Tre biosynthesis pathway, such as T6P,
trehalose and their biosynthetic enzymes are part of an interactive
correlation network including sugar and hormone signaling pathways,
etc[[43]7]. In plants, the components of Tre biosynthesis pathway not
only influence growth and development, but also get involved in
responses of both abiotic and biotic stresses[[44]7–[45]9].
Over-expressing yeast TPS1 and TPS2 in Arabidopsis can increase the
resistances of the transgenic lines to abiotic stresses, including
freezing, drought, salt and heat stress[[46]10]. The TPS1 transformants
of sorghum exhibited tolerance to salt stress as well as higher root
growth and biomass[[47]11]. In rice, over-expression of OsTPP1 confers
rice tolerance to both salt and cold stresses[[48]12], and OsTPP7 was
found as the genetic determinant in a major quantitative trait locus
(QTL) for an aerobic germination tolerance[[49]13]. Tre accumulated in
Tripogonloliiformis can regulate autophagy that might further confer
the plant desiccation tolerance[[50]14].
Tre is one of naturally occurring substances produced by organisms,
which is nontoxic to the environment. It showed elicitor and priming
properties, and improved protection in plants against abiotic and
biotic stresses.In wheat, exogenous Tre increases the resistance to the
biotic stress caused by powdery mildew[[51]15, [52]16]. In rice
seedlings, Tre pretreatment is involved in protection against
salt-induced oxidative damage through significantly enhanced level of
antioxidant activity[[53]17], and also elevated the endogenous Tre
level and significantly withstood the toxicities of excessive copper on
plant photosynthesis and development[[54]18]. Under drought stress, a
significant correlation has been found between exogenous application of
Tre and drought tolerance, and foliar spray of Tre was the best way in
improving antioxidant defense system in Raphanus sativus L. (radish)
plants[[55]19, [56]20]. A couple of studies have been done to
investigate gene expression patterns in response to Tre treatment in
Arabidopsis seedling cultured in liquid medium by DNA
microarray[[57]21, [58]22]. In tobacco, interestingly, exogenous Tre
can effectively increase nitrogen metabolism, and promote tobacco
growth under deficient nitrogen that restrict plant growth
severely[[59]23].
N. tabacum is the chief commercial crop among more than 70 species of
tobacco known. Tobacco mosaic disease (TMV) is one of the major
diseases of flue-cured tobacco. Studies have shown that pretreatment
with exogenous Tre can enhance plant's tolerance to both abiotic and
biotic stresses. However, whether Tre can enhance tobacco resistance to
TMV has not yet been documented. In the present study, we found that
exogenous Tre can enhance resistance of tobacco leaves to TMV in a
concentration dependent way. To elucidate the molecular mechanism of
the tobacco leaf in response to exogenous Tre treatment, firstly, the
best concentrations and time-points of exogenous Tre treatment were
determined by measuring a series of physiological responses of tobacco
leaves to exogenous Tre, and combined with the resistant efficiency of
tobacco to TMV affected by the different concentrations of Tre;
secondly, the transcriptomic responses of tobacco treated with Tre were
investigated using RNA-seq analysis. Our research provides a critical
basis for understanding the precise mechanisms that occur in leaf
tissues of the commercial crop N. tabacum in response to exogenous Tre
treatment.
Materials and methods
Plant material, treatment with exogeneous Tre and inoculation with TMV
Tobacco seeds (Nicotiana tabaccum cv. k326 and Nicotiana glutinosa. L.)
were germinated in petri dishes moistened filter paper. After 10 days,
seedlings were transferred in compost (Petersfield Products, Leicester,
UK) with no any other fertilizers in plastic pots (4 × 4 × 5 cm); 4–5
weeks later, plants were transferred to bigger plastic pots (diameter
20 cm; height 20 cm) containing the same compost. All plants used in
this study were grown in a constant temperature greenhouse at 24°C with
> 70% ambient humidity and 16 h light daily. Average photosynthetic
photon flux density of 300μmol (photon) m^−2 s^−1 at the height of
leaves employed for experiments. Tre treatment was tested for its
activity of increasing N. glutinosa resistance against TMV using the
half-leaf method (Necrotic local lesion assay in tobacco leaves by
means of half leaves). Tre solutions (Amresco, USA) of 10 and 50 mM
were freshly prepared before use in distillated water added with 0.025%
(v/v) Citowett, a wetting agent, whereas the corresponding control
contained distillated water with 0.025% (v/v) Citowett only. When
tobacco had eight fully expanded true leaves healthily at the 8 leaves
stage, leaves of the same age on different plants were selected for Tre
treatment, usually they were the sixth and the seventh. Half of the
whole leaf on both adaxial and abaxial surface was smeared 1 ml of
distillated water as a control, and the other half was done with 1 ml
of 10 mM or 50 mM Tre, respectively. For inoculation of TMV, the
adaxial surface of each leaf was gently rubbed with 100μl of TMV in
10mM phosphate buffer (pH 7.0) at concentration of 10 μg/ml and
Carborundum (silicon carbite) at 24h after Tre pretreatments. Numbers
of local lesions on each half leaf were noted at 4 days after TMV
inoculation. Fifteen replicates were performed for each Tre treatment
and control. The rate of inhibition was calculated in accordance to the
formula as follow:
[MATH: Inhibitionrate(%)
=Lesionnumberofcontrol–LesionnumberofTretreatmentLesionnumberofcontrol×100 :MATH]
Statistical data
To compare values from the raw data-number of lesions on each leaf
infected by TMV and stomatal apertures of tobacco leaves treated with
different concentration Tre, ANOVA was performed with the statistical
software DPS Data Processing System software V15.10 [[60]24]. The
Student-Newman-Keuls (SNK) (P< 0.05) method was used to discriminate
among the means.
Histochemical detection of O[2]^•- and H[2]O[2] accumulation in tobacco leaf
The histochemical detection of H[2]O[2] and O[2]^•-in tobacco leaf was
performedaccording to Hernandez et al. [[61]25] with minor
modifications. NBT staining was used to detect in situ the production
of O[2]^•-. 25 mM K-HEPES
[4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid] buffer (pH 7.6)
containing 0.1 mg mL^-1 NBT was infiltrated into tobacco leaves
directly by a vaccum, and incubated for 2 h at 25°C in the dark.
Tobacco leaf was then rinsed for 10 min with 80% (v/v) alcohol at 70°C,
and subsequently mounted in lactic acid/phenol/water [1:1:1, (v/v)] to
eliminate the chlorophyllcompletely, andphotographed directly using
camera.
In the case of H[2]O[2], DAB (3,3ʹ-Diaminobenzidine) stainingwas used
to detect in situ the production of H[2]O[2]. 50 mm Tris-acetate buffer
(pH 5.0) containing 0.1 mg/mlDAB was infiltrated intotobacco leavesas
above that were immersed into the same buffer for 24 h in the dark at
25°C. Controls were carried out and immersedinto the presence of 10 mm
ascorbic acid.
RNA-seq library preparation and illumina sequencing
Tobacco total RNAs from the leaf samples with Tre pretreatment were
extracted using RNeasy Plant Mini Kit (Qiagen GmbH, Gemany) as stated
in the manufacturer’s protocol. Firstly, RNA integrity and
contamination was assessedin 1.0% agarose gel by electrophoresis.
Further,the quality and concentration ofeach RNA sample were
analyzedtaking advantage of an Agilent 2100 Bioanalyzer. The NEBNext
Ultra RNA Library Prep Kit for Illumina (NEB, USA) was used for
generation of sequencing librariesaccording to manufacturer’s
recommendations, and the Agilent Bioanalyzer 2100 system was used
fordetermination of library quality, which were then sequenced to
generate 150 bp paired-end reads by 1 Gene Co. Ltd, Hangzhou, China
using the Illumina HiSeq 4000 Platform.
Functional annotation and enrichment pathway analysis of DEGs
In order to conduct the analysis of the raw RNA-Seq data, clean data
(clean reads) were obtained by removing low quality reads (reads
containing >20% of bases having Q value ≤15 or an ambiguous sequence
content (ploy-N) more than 5% from raw data) by Trimmomatic (v0.30)
[[62]26]. The reference of tobacco genome and annotation files of gene
model was obtained from the tobacco genome website to process the clean
data with high quality
([63]ftp://ftp.solgenomics.net/genomes/Nicotiana_tabacum/). Index of
the reference genome was built using Bowtie2 (2.3.0) [[64]27], and
subsequently, the reads were blasted to the reference genome of tobacco
using TopHat2 [[65]28].
The gene transcript abundance in all 12 samples was calculated as
fragments per kilo bases per million reads (FPKM) values[[66]29]. The
genes with FPKM<1 were filtered out before subsequent analysis.
Differential expression of two conditions was analyzed using DESeq
[[67]30] that supplies statistical principles for assessing
differential expression in RNA-seq data based on the negative binomial
distribution. The Benjamini-Hochberg adjusted P values were used for
controlling the false discovery rate (FDR). Gene with a very stringent
cutoff, an adjusted FDR <0.05 and |log[2]Ratio|≥1 identified by DESeq,
was classified as differentially expressed.
The Gene Ontology (GO) enrichment analyses of DEGs were performed using
the topGO R package with an adjusted P-valueless less than 0.05
([68]http://www.geneontology.org/). EdgeR package was used for testing
the statistical enrichment of DEGs in KEGG pathways
([69]http://www.genome.jp/kegg/).
qRT-PCR analysis for the DEGs
Tobacco total RNAs from leaf tissues sampledafter exogenous
Trepretreatment were extracted using the RNAiso Plus kit with DNase I
(Takara, Japan). First-strand cDNA was generated withthe oligo (dT)
prime using the PrimeScript OneStep RT-PCR Kit Ver. 2 (Takara, RR055A).
qRT-PCR experiment was performed usingthe CFX96 Touch™ Real-Time PCR
Detection System (Bio-Rad Laboratories, Inc., USA) with the SYBR Green
PCR Master Mix kit (PE-Applied Biosystems, USA).
Results
Pretreatment with exogenous Tre enhances resistance of tobacco leaf to TMV
infection significantly
In this study, we took advantage of the half-leaf method to determine
the optimal Tre concentration against TMV, We found were that both of
10 and 50 mM of Tre treatment before TMV infection can enhance
resistance of tobacco to TMV infection significantly in the light of
the numbers of local lesions on the leaves of N. glutinosa ([70]Table 1
and [71]S1 Data). The inhibition rates from 10 and 50 mM of Tre
pretreatments against TMV were 17.1% and 36.23%, respectively, which
are positively correlated with the concentration of exogenous Tre. Tre
treatment during and after TMV infection also showed protection
effects, butwas less efficient than a treatment before infection based
on our preliminary results (data not shown).
Table 1. Protection of trehalose against TMV.
Trehalose/mM Lesion number Inhibition rate/%
Control The leaves with trehalose
10 737 611 17.1
50 470 36.23
[72]Open in a new tab
The exogenous Tre as an effective elicitor increases superoxide anion
(O[2]^•-) and hydrogen peroxide (H[2]O[2]), reduces stomata aperture and
activates the expression of rbohD/rbohF
Combined with the effect of Tre concentration on inhibition rate of TMV
infection, a series of physiological phenotypes of tobacco leaves in
response to exogenous Tre were characterized to identify the optimal
time-points of Tre treatment for RNA-seq library construction. The
sixth and seventh fully expanded true leaves of N. tabaccum at the 8
leaves stage were smeared with 2 ml of 0 (as a control), 10 and 50 mM
of Tre on both sides, respectively, and the ROS species, O[2]^•- and
H[2]O[2], were monitored at 2h and 24 h after pretreatment separately
as described in methods. NBT staining and DAB staining indicated that
the production of both H[2]O[2] and O[2]^•-were significantly increased
in tobacco leaves at 2 and 24 h after Tre treatment ([73]Fig 1A). The
stomatal aperture was also measured at 2h after the treatments with 0,
10, 30, and 50 mM Tre, respectively. After Tre treatment, the stomatal
aperture of tobacco decreased significantly, and the percentage
decreases were 23.9%, 30.9% and 33.4%, respectively ([74]Fig 1B).
Fig 1. ROS content, stomatal aperture and enzyme expression profiles of
tobacco leaves in response to exogenous trehalose.
[75]Fig 1
[76]Open in a new tab
(A)Both superoxide ion (O^•-[2]) and hydrogen peroxide (H[2]O[2]) were
induced significantly at 2h and 24h after trehalose treatment; The
effect of trehalose on stomatal aperture without (B) (n = 48) or with
(C) (n≥22) CAT, imidazole and DPI;(D) The effect of trehalose on the
transcript level of rbohD and rbohF.
NADPH oxidases are generally membrane bound, and also named as
respiratory burst oxidase homologues (RBOHs) that catalyze the
reduction of molecular oxygen into O[2]^•- by transferring an electron,
wherein NADPH acts as an electron donor and are the biological ROS
factory and play a key role in the production of ROS in response to
both abiotic and biotic stress in plants[[77]31–[78]33]. Based on that,
some of the tobacco RBOHs might be activated by exogenous Tre, which is
likely to be due to the induction of the stomata movement and the
production of ROS in tobacco leaf. To test whether the tobacco RBOHs
are involved in that procedure, we treated the tobacco leaves with 50
mM Tre combined with catalase (CAT) (Sigma-Aldrich, cat#: C9322),
imidazole and DPI, respectively, as shown in [79]Fig 1C. CAT is a
catalase that clears H[2]O[2] from cells. Imidazole and DPI, as
inhibitor of NADPH oxidase, can bind to cytochrome B and flavoprotein
in NADPH oxidase to hamper ROS production. Exogenous CAT treatment had
little effect on Tre-induced reduction of stomatal aperture, while
imidazole and DPI treatment blocked Tre-induced stomatal closure and
returned to control levels of 95.02% and 94.52%, respectively ([80]Fig
1C). In N. tabacum, three members of NADPH oxidases/RBOHs have been
reported, and NtrbohD and NtrbohF are responsible for ROS
production[[81]34–[82]36]. The expression profiles of NtrbohD and
NtrbohF in response to exogenous Tre were determined by qRT-PCR.
NtrbohD was up-regulated only by the treatment of 10 mM Tre at 2 and
24h significantly, however, NtrbohF was up-regulated in all the four
conditions (10T2h, 50T2h, 10T24h and 50T24h) ([83]Fig 1D).
Sequencing overview and transcript identification
To investigate the molecular mechanism of the elicitor effect of
exogenous Tre,cDNA samples were sequenced using the Illumina HiSeq 4000
platform. According to the resistance of tobacco leaves with Tre
treatment to TMV infection and the physiological phenotypes above, the
leaves treated with 10 (low concentration) and 50 (high concentration)
mM of Tre were collected at two time-points, 2 (early stage) and 24h
(late stage) for RNA-seq analysis with two replicates with a total of
12 samples, including controls and four different conditions/groups
(combinations of two concentrations and two time-points). To simplify
the description, we designated each two replicates of the tobacco
leaves with 0 (control), 10 and 50 mM of T treatment for 2h or 24h as
CT2h-R1 and -R2, CT24h-R1 and -R2, 10T2h-R1 and -R2, 50T2h-R1 and -R2,
10T24h-R1 and -R2, and 50T24h-R1 and -R2, respectively. Therefore, the
four conditions/groups are named as 10T2h, 50T2h, 10T24h and 50T24h,
respectively.
Sequencing results showed that more than 45 million reads for each
sample were generatedfrom the 12 tobacco RNA libraries after the
Illumina HiSeq 4000 sequencing ([84]Table 2). Low-quality rRNA reads
and adapters were removed, leading to theratio ofclean readsmore than
91% for each sample, which denotes that we had the sufficient
sequencing depth for the transcriptome coverage in tobacco ([85]Table
2). Observed percentages of reads mapped to the N.tabaccum genome per
library were 79.4, 81.0, 86.1, 86.0, 86.2, 90.1, 82.6, 86.7, 85.7,
87.4, 83.6and 87.2%,respectively([86]Table 2), implying the RNA-seq
data are sufficient and reliable for following analysis of
bioinformatics.
Table 2. Summary of reads mapped to the N.tabaccum genome.
Sample ID Total Reads Clean Reads Total Mapped Reads MappedPairReads
Mapped Genes Q20 (%) GC (%)
10T2h_1 46,008,894 91.98% 79.4% 69.6% 36882 98.76 44.05
10T2h_2 45,054,250 92.47% 81.0% 69.2% 36941 98.82 44.02
10T24h_1 45,591,406 93.05% 86.1% 76.4% 37746 98.74 44.30
10T24h_2 48,229,638 92.84% 86.0% 75.6% 38065 98.69 44.13
50T2h_1 47,315,538 93.37% 86.2% 75.6% 37745 98.69 44.20
50T2h_2 45,769,886 94.83% 90.1% 81.1% 37724 98.69 43.91
50T24h_1 47,589,142 93.66% 82.6% 70.6% 37530 98.75 43.91
50T24h_2 48,076,528 92.68% 86.7% 76.2% 37449 98.66 44.02
C2h_1 47,337,734 96.51% 85.7% 77.4% 37947 98.82 44.05
C2h_2 46,996,730 94.57% 87.4% 78.3% 37615 98.75 44.02
C24h_1 45,862,902 93.35% 83.6% 75.3% 37486 98.86 44.30
C24h_2 46,758,318 94.31% 87.2% 78.7% 37807 98.81 44.13
[87]Open in a new tab
DEGs responding to exogenous Tre and qRT-PCR validation
As shown in [88]Table 3, in the four comparisons of 10T2hvs C2h,
50T2hvs C2h, 10T24h vs C24h and 50T24hvsC24h, there were 242, 102, 833
and 111 DEGs indentified by DESeq, respectively. The maximum number of
DEGs was in 10T24h vs C24h, which indicates low concentration of T
(10mM) at late stage (24h) triggers more DEGs than the other three
conditions. 110, 64, 579 and 56 were up-regulated, while 132, 38, 254
and 55 were down-regulated in the 10T2hvs C2h, 50T2hvs C2h, 10T24hvs
C24h and 50T24h, respectively ([89]Table 3).
Table 3. The DEGs in tobacco leaves responding to trehalose compared with
control.
Group Total UP Down
10T2h vs C2h 242 110 132
50T2h vs C2h 102 64 38
10T24h vs C24h 833 579 254
50T24h vs C24h 111 56 55
[90]Open in a new tab
To experimentally confirm that the DEGs obtained in this study were
credible, expression profile of 19 selected DEGs, 2 (FSH and HSP21) of
them up-regulated in all the four groups, were analyzed via qRT-PCR
([91]Fig 2). The tobacco Actin2 (GenBank accession No.[92]EU938079.1)
was chosen as a reference gene for qRT-PCRassay, and the2^-ΔΔCT method
was used for calculation of relative expression levels of DEGs that
were normalized to reference gene Actin2 [[93]37]. Three biological
samples were prepared for each condition, and six conditions were
included in [94]Table 3. Three whole leaves except for main veins were
collected as one independent biological sample. Two technical
replicates for each sample were used for each DEG selected. The qPCR-RT
results showed that the expression profiles of the DEGs selected were
in line with those obtained from the Illumina sequencing analysis. As
shown in [95]Fig 2, comparison of qRT-PCR results with RNA-seq data
showed high correlations (R2≥0.792 for each condition/group),
confirming accountable RNA-seq analysis in the present study. These
results indicated that the method used to determine DEGs in this study
were valid. The primers used in qRT-PCR analysis were shown in [96]S2
Data.
Fig 2. qRT-PCR validation of the DEGs selected from the four conditions
(10T2h, 50T2h, 10T24h and 50T24h).
[97]Fig 2
[98]Open in a new tab
The expression profiles were compared with the corresponding DEGs in
RNA-seq data (R^2 = 0.792, R^2 = 0.873, R^2 = 0.917, R^2 = 0.871),
respectively. R represents correlation coefficient.
A total of 1288 DEGs from all four conditions were further assorted by
Venn analysis ([99]Fig 3 and [100]S3–[101]S6 Data). The Numbers
indicate unique and common DEGs for the different comparisons in the
Venn diagram. From it, what we found was Tre affected the gene
expressions of tobacco leaves in both a concentration-dependent manner
and a time-specific manner. 929 (in response to 10 mM Tre) and 96 (in
response to 50 mM Tre) DEGs regulated by effect of Tre concentration
uniquely were confirmed, respectively ([102]Fig 3, [103]S7 and [104]S8
Data), indicating that low concentration of Tre (10 mM) triggered much
more genes expressed differentially than high concentration of Tre (50
mM) at both 2h and 24 h, and that was a much broader and more expensive
response to 10 mM Tre. 160 unique DEGs were in 10T2h. Among them, 62
were up-regulated, and 98 were down-regulated. The majority of the DEGs
regulated by 10 mM Tre were from 10T24h. Among them, 539 were
up-regulated, while 215 were down-regulated. The DEGs also exhibited a
time-specific expression pattern, a shift of numbers of DEGs from 2h to
24h was observed. Therefore, we compared and analyzed the DEGs in these
two aspects.
Fig 3. Venn diagram of the DEGs in different comparisons from the four
conditions including 10T2h, 10T24h, 50T2h and 50T24h.
Fig 3
[105]Open in a new tab
The Numbers (Red: up-regulated genes; Black: down-regulated genes)
indicate unique and common DEGs for the different comparisons.
Functional classifications of the DEGs in response to Tre in a
concentration-dependent manner by GO and KEGG pathway enrichment analysis
To uncover the similarities and differences of biological procedures in
Tre-responsive transcriptomes between controls and the four different
conditions, the 1288 DEGs from all four conditions were annotated and
mapped to GO term and KEGG database, respectively. The Tre
concentration comparison was performed in terms of the low (10mM) to
high (50mM) concentration of Tre. Based on sequence homology, they were
assigned to three ontologies of biological process (BP), cellular
component (CC) and molecular function (MF) ([106]Fig 4; [107]S7 and
[108]S8 Data). The significant GO terms were very similar between the
DEGs triggered by 10 mM Tre and 50 mM of Tre, and were mostly enriched
in metabolic process, cellular process, single-organism process, and
respond to stimulus in the ontology of BP. Most of the DEGs in the CC
category were assigned to cell, cell part, membrane, organelle and
membrane part. The top three GO terms in the MF category were catalytic
activity, binding and transporter activity. The corresponding genes of
these significant terms, therefore, might play important roles in
response to abiotic and biotic stresses.
Fig 4. The comparison of the GO terms for the DEGs triggered by 10 mM and 50
mM of trehalose, respectively.
[109]Fig 4
[110]Open in a new tab
The DEGs from 10 mM and 50 mM of Tre treatment also had similar pattern
in KEGG analysis. The 1288 total identified DEGs were assigned to 78
KEGG pathways ([111]Fig 5, [112]S7 and [113]S8 Data) that were
represented in [114]Fig 5, and were largely enriched in biotic and
abiotic stresses belong to metabolic pathway, biosynthesis of secondary
metabolites, plant hormone signal transduction, plant-pathogen
interaction, starch and sucrose metabolism, protein processing in ER,
flavonoid biosynthesis, phenylpropanoid biosynthesis and pentose and
glucuronate interconversion, etc. These results indicate that exogenous
Tre can trigger a lot of stress-related genes in tobacco leaves, which
might be involved in partial resistance to TMV. However, the amount of
the DEGs in response to low concentration of Tre (10mM) were much more
than those regulated by high concentration of Tre (50mM), their
patterns were similar in different pathways though. For example, 173
DEGs triggered by 10 mM of Tre were enriched in metabolic pathway, in
contrast, only 26 DEGs regulated by 50 mM of Tre treatment were found
in the same pathway. However, in protein processing in ER, the amount
(29) of DEGs triggered by 10 mM of Tre was similar to those (22)
triggered by 50 mM of Tre. The overlap between them included 9 genes
that were all increased in response to both of 10 and 50 mM of Tre
treatment and encode heat shock proteins ([115]Fig 5, [116]S7 and
[117]S8 Data), which suggests that the concentration of exogenous Tre
has a strong effect to the gene expression profiles of tobacco leaves.
Fig 5. The Comparison of KEGG classification for the DEGs regulated by 10 mM
and 50 mM of trehalose, respectively.
[118]Fig 5
[119]Open in a new tab
DEGs independent of Tre concentration in tobacco leaves relative to
time-specific expression pattern
We further compared the differences and similarities of DEGs during the
progression of Tre treatment as they might show the putative roles
involved in resistance or susceptibility to abiotic and biotic
stresses. At early time point (2h), 49 genes respond to Tre treatments
free from effect of Tre concentration, in which 31 were up-regulated
and 18 were down-regulated, respectively. At the later time point
(24h), 35 genes respond to Tre treatments independent of effect of Tre
concentration, where 12 were increased and 23 were decreased,
respectively ([120]Fig 3). Among these genes, 8 genes, named as core
components in response to exogenous Tre, exhibited regulatory responses
to all the four conditions, and were all up-regulated, including four
heat shock protein (HSPs) genes (HSP101, HSP90, HSP70T-2 and HSP21),
two FTHS6s, one GolS1and one universal stress protein (USP) gene
([121]S9 Data).
To elucidate the complex interaction of the differentially expressed
proteins independent of effect of Tre concentration at each time point,
the TAIR Arabidopsis gene codes of the DEGs were obtained via BLASTx
([122]S10 and [123]S11 Data), and subsequently imputed into STRING tool
([124]www.string-db.org) to view protein-protein interaction networks
(PPINs). The network of time point 2h indicates that Tre triggered a
heat shock-like response at early stage ([125]Fig 6A). Majority (31/49)
of the DEGs were increased significantly, including the 8 core DEGs
([126]S9 Data). Many of them, such as APX2, GolS1, HSPs, etc., are the
targets of some key HSFs, like HSFA1 and HSFA2. HSFA2 might be
responsible for their activations in tobacco leaves with Tre treatment
since it has been reported that the peak of HSFA2 expression is around
0.5-1h under heat stress [[127]38, [128]39], which can explain HSFA2
was not up-regulated significantly at 2h after Tre treatment in our
RNA-seq data. In addition, jasmonate signaling pathway involved plant
defense and diverse developmental pathways was clustered in PPIN of the
2h stage ([129]Fig 6A), suggesting that Tre treatment can activate the
pathway at early stage effectively. Down-regulation of
JasmonateZim-domain protein 1(JAZ1) were observed at 2h stage in both
conditions of 10T2h and 50T2h. JAZ proteins as transcription repressors
bind to promoter regions of jasmonate-inducible genes to block their
expression [[130]40].
Fig 6. STRING network of trehalose-responsive genes at each time point (2h
and 24h).
Fig 6
[131]Open in a new tab
In the network of time point 24h ([132]Fig 6B and [133]S10 Data),
HSFB2B with some HSPs/chaperones were clustered. HSFB2B represses the
transcription of heat shock response genes under normal temperature
conditions. In contrast, under heat stress conditions, it is required
for the transcription of heat stress-inducibleHSP genes that are
indispensable for acquired thermo-tolerance [[134]41]. The expression
of HSFB2B is necessary to attenuate harmful effect of abiotic stress on
the circadian system [[135]42], and it is also involved in resistance
to biotic stress in Arabidopsis [[136]43]. Therefore, the DEGs from the
RNA-seq data constructed a strong connection between exogenous Tre and
the signal pathways of both abiotic and biotic stresses.
Discussion
Pretreating crops with natural isolated elicitors in the absence of
pathogens can enhance plant resistance to diseases. Up to now, diverse
elicitors are available from organisms such as bacteria, fungus,
oomycete and plants. Nonetheless, different elicitor usually does not
trigger same plant response. Therefore, their molecular networks need
to be elucidated prior to the application of elicitor for good
agriculture practice. Tre is a non-toxic disaccharide synthesized by
organisms such as some bacteria, fungi, plants and invertebrate
animals, and is a good candidate of elicitor against environmental
stresses, especially plant diseases, as it has been more affordable and
accessible due to its chemical synthesis and, when applied exogenously,
is readily absorbed by plants [[137]44]. Evidences showed that
exogenous Tre reduced stomata aperture through a H[2]O[2]-dependent
pathway [[138]45, [139]46], and stomata movement in response to abiotic
and biotic stress are regulated by redox-dependent signaling [[140]47,
[141]48]. It has been used as a tool to enhance plant resistances to
some of abiotic and biotic stresses that affect plant growth and yield.
However, exogenous Tre is more or less toxic to plant development. In
Arabidopsis, 100 mM of exogenous Tre treatment results in strong
inhibition of seedling growth. T6P rapidly accumulating in cytosol
might mediate the signal transduction pathways of growth inhibition
caused by Tre [[142]49], which enhances starch biosynthesis and
triggers redox activation of ADP-glucose pyrophosphorylase (AGPase)
gene in source tissues[[143]50]. Microarray analysis shown that
Arabidopsis seedlings treated with 30 mM of had many changes on its
transcripts related to metabolism, abiotic and biotic stresses, and
root elongation was also inhibited significantly [[144]21]. However,
low millimolar concentrations of exogenous Tre can attenuate impairment
of salt stress to Arabidopsis [[145]51] and Catharanthus [[146]52]
through regulation of ionic balance, cellular redox state, cell death,
and osmotic adjustment. During the process, Tre toxicity to plant was
counteracted by impairment of salt stress. Furthermore, in tobacco, at
low concentration (e.g. 8 mM), exogenous Tre can recover the nitrate
reductase activity partially, and chlorophyll and total nitrogen
content of leaves and rates of photosynthesis were increased through a
long-term effect, which can be observed 1–2 weeks after the treatments
[[147]23]. In stark contrast, we did not find the expression level of
any nitrate reductase genes were changed in our RNA-seq data ([148]S7
and [149]S8 Data). One possibility is we only focused on the molecular
mechanism occurring early (in 24 h) and without any environmental
stresses; and nitrate reductase genes are uniquely affected by Tre
under limiting nitrogen condition. The interesting differences between
our present research and Lin et al’s work [[150]23] suggest that there
might be some other mechanisms of plant responding to exogenous Tre
treatment, such as performing the treatments during or after
environmental stresses.Similar to this, in the present study, high
concentration of Tre (50 mM) induced less number of DEGs, but showed a
better protection against TMV. One possibility is, as a natural
chemical compound, high concentration of Tre might act mainly as a
priming agent, but not an elicitor, which means Tre can act mainly as
an elicitor or a priming agent in one species in a
concentration-dependent way, because it has been reported one compound
can function as an elicitor or a priming agent between different
species [[151]53].
Plant pathogen is perceived by plant surface pattern recognition
receptors that detect conserved microbe-specific molecules referred as
to pathogen-associated molecular patterns (PAMPs). Flagellin
insensitive 2 (FLS2), EF-Tu receptor (EFR) and chitin elicitor receptor
kinase 1 (CERK1) are this sort of receptors that have been
characterized recently referred as to pattern-recognition receptors
(PRRs) involved in the specific interaction with corresponding
elicitors, respectively [[152]54–[153]56]. The three receptors,
BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) and BAK1-LIKE1
(BKK1) functioning as the partners of FLS2 and EFR, were all increased
significantly and uniquely at 24h following 10 mM of Tre treatment
([154]S13 Data; [155]S5 Fig), which are strongly indicating that low
concentration of exogenous Tre can effectively trigger plant-pathogen
interaction pathways in tobacco leaf in a time-specific way. Activation
of these receptors and their partners lead to changes in ion flux and
metabolism, accumulation of ROS and hormone ethylene and MAP kinase
(MPK) activation, etc [[156]57]. Based on our RNA-seq data, the
Plant-pathogen interaction pathways were enriched in both 10T2h and
10T24h. 20 DEGs from 10T2h and 61 DEGs from 10T24h were mapped to this
pathway, respectively ([157]S4 and [158]S5 Figs). The majority (15/20 =
75%) of the 20 genes in 10T2h ([159]S12 Data) were down-regulated. In
contrast, 53/61 (around 87%) in 10T24h ([160]S13 Data) were
up-regulated, indicating that the activation of pathogen resistance
genes by exogenous Tre is time-dependent. Beside those receptor like
kinases mentioned above, Some cyclic nucleotide gated channels (CNGCs)
proteins sub-locating on cytoplasmic membrane as the upstream
components in the pathways were up-regulated significantly in the
condition of 10T24h. CNGCs activates downstream of Ca2+ -mediated
signal ([161]S5 Fig; [162]S13 Data). Functional analyses of members of
this channel family have associated many of them with inward Ca^2+
currents [[163]58]. More and more evidence implies there is a mutual
interplay between Ca^2+- and ROS-mediated signaling pathways that might
be involved in fine-tuning of intercellular and intracellular signaling
networks [[164]59]. One example is calcium acts as the prime regulatory
molecule of NADPH oxidases/RBOHs that are the key player of pathogenic
ROS generation in plants [[165]60, [166]61]. In the model plant
Arabidopsis thaliana, there are 10 members of RBOHs whereas nine
members are present in rice plants [[167]61]. In Nicotiana tabacum
three members are reported [[168]34, [169]35]. We measured the mRNA
levels of rbohF, rbohD by qRT-PCR ([170]Fig 1D), since they two have
been proved for ROS generation in plants [[171]34–[172]36], and both
superoxide ion and hydrogen peroxide were induced significantly at 2h
and 24h after Tre treatment in tobacco leaves ([173]Fig 1A). One RBOH
gene in the RNA-seq data (gi|697111405|) was increased significantly at
24h after 10 mM of Tre treatment too ([174]S5 Fig and [175]S13 Data).
ROS are high reactive molecules, and their excessive accumulation might
lead to strong damages to cell membrane system and other structures,
thus originally they were deemed as detrimental byproducts during
aerobic metabolism, especially when organism suffers environmental
stresses. Now, it is undoubted that ROS of sub-toxic levels function as
signaling molecules sensing diverse upstream signals coming from
developmental processes and environmental stresses [[176]59, [177]62],
and subsequently transduce the signals to downstream targets as a
common plant response. Different environmental stresses or stress
combinations that occur in field condition often might lead to the
formation of different ROS signatures, which perceived by diverse ROS
sensors that activate the corresponding stress signals in plants
[[178]63–[179]65]. For instance ([180]S5 Fig), Ca^2+ and ROS induce
expression of pathogenesis-related (PR) genes and formation of
localized cell death (LCD) at the site of infection (hypersensitive
response) through activation of CDPK that was up-regulated
significantly in the condition 10T24h ([181]S13 Data); Ca^2+ induces
stomatal closure appears through calmodulin (CaM) and calmodulin-like
(CML) proteins (main calcium sensors)and ROS/NO productions [[182]66,
[183]67]. In this study, up-regulation of CaM/CML was observed in the
condition 10T24h ([184]S5 Fig and [185]S13 Data), and ROS
(O[2]^•-andH[2]O[2]) were accumulated in tobacco leaves after Tre
treatment ([186]Fig 1A). WRKY transcription factors, including WRKY22,
25, 29 and 33, were up-regulated in the condition 10T24h ([187]S13
Data), and they are downstream of mitogen-activated protein kinases,
MPK3/6 and MPK4. MPK4 can sense Ca^2+-mediated signals and activate the
WRKY transcription factors ([188]S5 Fig). Although we did not observe
any MPK3, MPK4 and MPK6-like genes in the 1128 DEGs, gene_23843 encodes
AP2C1 protein which belongs to the PP2C-superfamily clade B and
functions as a MAPK phosphatase that negatively regulates MPK4 and MPK6
[[189]68]. In the RNA-seq data, AP2C1 was down-regulated significantly
at 2h after Tre treatment independent of its concentration ([190]S10
Data and [191]Fig 6A), suggesting some MPKs might be activated during a
particular window after Tre treatment.
HSPs, also known as molecular chaperones, play critical roles in
protein correct folding and subunit assembly, translocation between
cellular compartments, and targeting misfolded proteins to the
proteasome in diverse normal cellular processes, and protect and
stabilize proteins and membrane system against environmental
stresses[[192]69, [193]70], and their expressions are usually regulated
by heat shock factors (HSFs) that also respond to diverse biotic and
abiotic stresses. For example, it has been reported that Arabidopsis
HSFA2, as a key regulator, is involved in signaling pathways of heat,
high light and osmotic stress [[194]38, [195]71]. Accumulated evidence
have been gotten recently on redox-dependent regulation of HSFs in
plants and mammalian [[196]72, [197]73], further supporting the
hypothesis that some oxidative stress-responsive genes are probably
direct targets of oxidative stress-responsive HSFs that act as H[2]O[2]
sensors in plants [[198]74]. It is evidenced by the report that
Arabidopsis HSFA1a directly senses H[2]O[2] via its N-terminal region
from 48 to 74 amino acid residues [[199]75]. FTHS6 encodes a plastid
metalloprotease, and it regulates thermomemory in Arabidopsis through
regulating HSP21 abundance [[200]76]. Moreover, down-regulation of
chloroplast FtsH Protein in TMV–infected tobacco leaves accelerates the
hypersensitive reaction [[201]77]. GolS1 encodes a galactinol synthase
and is a target of AtHSFA2 under heat stress [[202]38, [203]71]. The
transcription of AtGolS1 was also induced by exogenous H[2]O[2] in
Arabidopsis [[204]78] and other abiotic stresses such as drought, salt,
or heat stress [[205]79] and hormone ABA [[206]80]. Its over-expression
in transgenic plants promoted accumulation of galactinol, raffinose and
stachyose, which resulted in enhanced resistance to abiotic stresses
such as drought, salinity or cold [[207]79, [208]80]. It has been
reported that an Arabidopsis USP modulates ROS homeostasis under anoxic
conditions [[209]81], and one USP in tomato is a phosphorylation target
of protein kinase CIPK6 and functions in oxidative stress response
pathway [[210]82]. Therefore, the 8 core genes are all in response to
oxidative stress directly or indirectly, indicating that ROS are the
key regulators for the DEGs triggered by exogenous Tre.
There are some important common mediators such as calcium ions between
heat shock responses and defense responses [[211]83]. Our RNA-seq data
also evidenced there is a connection between components in
plant-pathogen interaction pathways and HSPs triggered by exogenous
Tre. As mentioned above, ROS can stimulate some HSFs directly. These
HSFs as ROS sensors in turn activate expression of HSP chaperone genes
and also regulate expression of ROS scavenger genes [[212]73], such as
APX2 that was up-regulated at 2h after Tre treatment ([213]Fig 6A),
which is responsible for keeping ROS homeostasis through negative
feedback. Many of HSFs can specifically bind DNA sequence
5’-AGAAnnTTCT-3’ referred to as heat shock promoter elements (HSE) that
are included in the promoter regions of HSPs. HSFA6B, HSFB2A and 3
HSFB2Bs were up-regulated in one or two conditions out of the four
conditions ([214]S3–[215]S6 and [216]S14 Data). Their orthologs in
Arabidopsis or rice have strong correlations with MPK3/6 and 4,
respectively, base on PPINs (STRING analysis) ([217]S1–[218]S3 Figs).
Plant pathogen signaling pathway in KEGG shows that MAP3/4/5/6 play
keys roles in the signal transductions of pathogen infection and/or
attack together or alone (S4 and [219]S5 Fig). HSFA6B has connections
with eight MPKs (3,4,5,6,7,10,12 and 14) and two HSP70 proteins
([220]S1 Fig). HSFB2A associates with four MPKs (1, 3, 5 and 11) and
some HSPs (e.g. HSP 70, 80 and 90) ([221]S2 Fig). Although in
Arabidopsis, AtHSFB2B only has second shell of interactions with MPK6
([222]S3A Fig); in rice, HSFB2B (rice gene ID: 4346226) has
correlations with three MPKs, respectively (rice gene ID: 4344698,
4341956 and 4349225) ([223]S3B Fig). It has been reported MAP kinase
kinase 5 (MPPK5) might be a direct target of HSFB1/HSFB2B based on
microarray analysis [[224]43]. In addition, HSFA4A confers plant salt
resistance and is regulated by oxidative stress. It has a physical
interaction with MPK3 and MPK6 and is phosphorylated by the two
mitogen-activated protein kinases [[225]84]. Arabidopsis HSFA2 is also
regulated by MPK6-targeted phosphorylation during heat stress response
[[226]85]. The heat shock transcription factor HSFB2B is strongly
responsive to photorespiratory H2O2 [[227]86]. Some HSPs as HSFs
targets are not only involved in heat stress, but also function in
microbial pathogenesis such as HSP70 [[228]87]. In addition, HSP90 gets
involved in innate immune responses in tobacco through interacting with
SGT1 and RAR1 they are critical signaling components in resistance (R)
gene-mediated plant resistance responses [[229]88], and HSP90 is also
involved in resistance to TMV through interacting with resistance
protein N [[230]88, [231]89]. Besides the top ten KEGG pathway in which
the total DEGs were mapped, we found that some other pathways were
uniquely enriched in the DEGs triggered by 10mM of Tre, but not in
those triggered by 50 mM of Tre, such as ATP-binding cassette
transporters (ABC transporters). In the RNA-seq data, 8 ABC transporter
genes were up-regulated and 1 gene was down-regulated ([232]Fig 5;
[233]S7 Data). The integral membrane proteins transporters are
widespread in all living organisms and can be grouped into exporters
and importers. They are not only required for organ growth, plant
nutrition and plant development, but also respond to both abiotic and
biotic stresses [[234]90].
In summary, exogenous Tre as an elicitor molecule recognized by tobacco
cells, and trigger intracellular pathways related to defense, via the
mutual interplay between calcium and ROS signaling systems ([235]Fig
7).
Fig 7. Core signaling in response to exogenous trehalose treatment.
[236]Fig 7
[237]Open in a new tab
Abbreviations:PRRs—Pattern-recognition receptors; FLS2—Flagellin
insensitive 2; EFR—EF-Tu receptor; CERK1—Chitin elicitor receptor
kinase 1;CNGCs—Cyclic nucleotide gated channels; O[2]^•-—superoxide
ion; H[2]O[2]—hydrogen peroxide; MPKs—MAP kinases; APX2—Ascorbate
peroxidase 2; HSFs—Heat shock factors; TF—Transcription factor;
HSPs—Heat shock proteins. Dotted arrows represent the unknown pathways.
Conclusion
This study provides new insight into the molecular basis of Nicotiana
tobacum leaves in response to exogenous Tre treatment by comprehensive
analysis of gene expression profiles. Our results also showed that
tobacco leaf treatment with higher concentration of Tre (50 mM)
conferred the plant better disease resistance to TMV. In contrast, a
lower concentration of Tre (10 mM) triggered more genes differentially
expressed, which are responsive to Ca^2+ and ROS-mediated signaling, in
noninfectious context. However we didn't investigate any priming effect
of Tre which could be involved in protection induced against TMV.
Priming is characterized by a faster and/or stronger activation of
cellular and molecular defenses responses that are induced following a
pathogenic attack. Such a priming effect might explain the better
protection obtained with 50mM Tre pretreatment before infection with
TMV. For further researches, we will investigate this possibility, and
characterize the molecular functions of the DEGs triggered by exogenous
Tre via transgenic approaches.
Supporting information
S1 Data. The statistical data of [238]Table 1.
(XLSX)
[239]Click here for additional data file.^ (14.3KB, xlsx)
S2 Data. List of primers for qRT-PCR.
(XLSX)
[240]Click here for additional data file.^ (11.2KB, xlsx)
S3 Data. DEGs expressed in the condition of 10T2h.
(XLSX)
[241]Click here for additional data file.^ (129.2KB, xlsx)
S4 Data. DEGs expressed in the condition of 10T24h.
(XLSX)
[242]Click here for additional data file.^ (600.8KB, xlsx)
S5 Data. DEGs expressed in the condition of 50T2h.
(XLSX)
[243]Click here for additional data file.^ (60.7KB, xlsx)
S6 Data. DEGs expressed in the condition of 50T24h.
(XLSX)
[244]Click here for additional data file.^ (61.4KB, xlsx)
S7 Data. DEGs regulated by 10 mM of trehalose uniquely.
(XLSX)
[245]Click here for additional data file.^ (178.4KB, xlsx)
S8 Data. DEGs regulated by 50 mM of trehalose uniquely.
(XLSX)
[246]Click here for additional data file.^ (51.8KB, xlsx)
S9 Data. Overlapping DEGs regulated by all conditions.
(XLSX)
[247]Click here for additional data file.^ (11.4KB, xlsx)
S10 Data. Overlapping DEGs regulated by 10T2h and 50T2h.
(XLSX)
[248]Click here for additional data file.^ (19.3KB, xlsx)
S11 Data. Overlapping DEGs regulated by 10T24h and 50T24h.
(XLSX)
[249]Click here for additional data file.^ (18.1KB, xlsx)
S12 Data. DEGs of 10T2h in plant pathogen pathway in KEGG.
(XLSX)
[250]Click here for additional data file.^ (11.6KB, xlsx)
S13 Data. DEGs of 10T24h in plant pathogen pathway in KEGG.
(XLSX)
[251]Click here for additional data file.^ (18.8KB, xlsx)
S14 Data. HSFs induced by exogenous trehalose.
(XLSX)
[252]Click here for additional data file.^ (9KB, xlsx)
S1 Fig. The PPIN of HSFA6B and MPKs.
(TIF)
[253]Click here for additional data file.^ (1MB, tif)
S2 Fig. The PPIN of HSFB2A and MPKs.
(TIF)
[254]Click here for additional data file.^ (8.7MB, tif)
S3 Fig. The PPIN of HSFB2B and MPKs.
(TIF)
[255]Click here for additional data file.^ (2.3MB, tif)
S4 Fig. KEGG map of 10T2h DEGs in plant pathogen pathway.
(TIF)
[256]Click here for additional data file.^ (493.2KB, tif)
S5 Fig. KEGG map of 10T24h DEGs in plant pathogen pathway.
(TIF)
[257]Click here for additional data file.^ (168.3KB, tif)
Data Availability
All relevant data are within the manuscript and its Supporting
Information files.
Funding Statement
HY received the National Natural Science Foundation of China
(31871368), which website is [258]http://www.nsfc.gov.cn/. The funder
had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript. None of the research costs
or authors' salaries have been funded in whole or in part by a tobacco
company.
References