Transcriptome Analysis to Elucidate the Enhanced Cold Resistance of Phoebe zhennan Pretreated with Exogenous
Calcium
Bo Deng, Botao Jia, Guihua Liu*
and Xiaoyan Zhang
School of Forestry and
Landscape Architecture, Anhui Agricultural University, Hefei 230036, P. R.
China
*For correspondence: liuguihua1968@163.com
Received
17 June 2020; Accepted 09 September 2020; Published 10 December 2020
Abstract
Phoebe zhennan is a high-quality timber tree species, and cold stress is one of the most
remarkable abiotic factors limiting its growth and development. In this study,
effects of exogenous CaCl2 on cold resistance of P. Zhennan were surveyed. CaCl2-pretreatment
increased the levels of abscisic acid, peroxidase, catalase, proline, and
soluble sugar, while decreased the levels of malondialdehyde and relative
electrical conductivity. In addition, RNA-sequencing was used to investigate
global transcriptome responses to cold stress. A total of 4245 differentially
expressed genes were identified, including 477 up-regulated and 3768
down-regulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
analysis indicated that these DEGs were associated to the plant-pathogen
interaction pathway, in which calcium-dependent protein kinase (CDPK) play key
role for improving the cold resistance of P.
zhennan. Our results can be useful to understand the Ca2+-mediated
cold resistance mechanism for improving cold stress tolerance in P. Zhennan seedlings. © 2021
Friends Science Publishers
Keywords: Phoebe
Zhennan; Calcium chloride; Cold stress;
Transcriptome profiling; Calcium-dependent protein kinase
Introduction
In natural environments or agricultural practices,
growth and production of plants were usually suppressed by environmental
stresses. Freeze injury caused by subzero temperature is one of the most common
stresses, especially for plants distributed in sub-tropical regions (Rooy et al. 2017). When plants suffered from
low temperature, various physiological processes can be activated, including
molecular and biochemical processes, and by altering gene expression level and
phenotype to adapt to low temperature. In previous studies, low temperature
down-regulated photosynthesis-related protein expression, sugar transport,
respiratory rate, while up-regulated carbohydrate metabolism, cell wall
remodeling, redox adjustment, and defence/detoxification (Janmohammadi et al. 2015; Ding et al. 2019; Leites et al.
2019). Abscisic acid (ABA) plays a very crucial role for improving plant cold
resistance (Fu et al. 2017). Protein
biosynthesis induced by low temperature was similar with ABA or light water
shortage, and these proteins possessed the functions of anti-freezing and
osmotic adjustment (Mantyla et al.
1995). It is now possible to investigate the response of plant to environmental
stresses on global transcriptional level with the development of
transcriptomics.
Phoebe zhennan, commonly called Zhennan, is an evergreen plant that belongs to the
Lauraceae family and is mainly distributed in southwest of China (Chen et al. 2018). Zhennan is a high-quality
timber tree species and is well known for its remarkable surface golden tint.
Since Ming Dynasty, the wood was widely used as the materials of palace
buildings, such as the Forbidden City (Xie et
al. 2015). This tree species is also characterized as an ornamental plant,
usually for landscaping (Hu et al.
2015). However, the extensive deforestation in recent centuries, the large
diameter Zhennan timbers are increasingly scarce. In the past few years, the
natural resources of Zhennan have been continuously declined (Cao et al. 2016; Gao et al. 2016). In the recent years, the conservation and recovering
of rare and precious tree species resources, including Zhennan, received more
attention (Hu et al. 2015). At
present, cultivation of Zhannan has gained attention for ecological restoration
and use in timber resource. To our knowledge, introduction and cultivation of
Zhennan has been carried out in southeast regions of China in the past few
decades. As a sub-tropical tree species, however, Zhennan usually meet with
freeze injury in winter when temperature below freezing.
Calcium ions (Ca2+), the
crucial second messengers, involve in mediating responses to many abiotic
stimuli, including low temperature, salinity and drought (Perochon et al. 2011; Wang et al. 2016). Previous studies found that the stress tolerance of
plants can be improved through the Ca2+ application. Huang et al. (2015) found that exogenous Ca2+
application enhanced the cold tolerance of tea plant. Trapnell et al. (2011) also found a post-drought
recovery phenomenon of tea plant by foliar-sprayed with Ca2+.
However, little information is available about the effects of exogenous Ca2+
application on cold tolerance of Zhennan. The present study aims at
investigating the physiological mechanisms of cold tolerance in Zhennan on
transcriptional and metabolic levels. The information would be of great value
for breeding and cultivation.
Materials and Methods
Plant materials and experimental design
The 1-year-old seedlings of Zhennan
were cultivated in a controlled phytotron with a 50% relative humidity, 12 h
photoperiod, and a 17°C/12°C diurnal/night temperature for 25 d. The leaves of
Zhennan seedlings were then continuously sprayed daily with 10 mmol/L CaCl2 solution
or distilled water for 7 d. On the eighth day, all seedlings were
moved to open field and exposed to natural low temperature condition. The first
leaves below the top buds were collected, and immediately frozen in liquid
nitrogen, and stored at -70℃ before and
after the cold treatment (i.e., 3 and 15 d). During the experimental
period, the daily air temperature was shown in Fig. 1. There were 6 seedlings
in treatments of CaCl2-pretreated and control and three biological
replicates in each treatment.
Measurements of physiological and biochemical
parameters
The leaves of Zhannan seedlings
were sampled according to each replication and the fresh leaves were used to
measure physiological and biochemical parameters. The levels of ABA, peroxidase
(POD), catalase (CAT) were measured based on the protocol of Plant ABA ELISA
Kit, Plant POD ELISA Kit and Plant CAT ELISA Kit, respectively. The contents of
chlorophyll were detected by using spectrophotometry at 663 and 645 nm,
respectively, after extracting with acetone-ethyl alcohol (v/v = 17/3) solution
(Paul and Driscoll 1997). Relative electrical conductivity was analyzed as a
method described by Cui et al.
(2018). The contents of proline, malondialdehyde (MDA), and soluble sugar were
measured according to a method described by Bates et al. (1973), Draper and Hadley (1990), Buysse and Merckx (1993),
respectively.
RNA
extraction, cDNA library construction, and RNA-sequencing
In order to perform the transcriptome sequencing
analysis, the leaves of Zhennan from CaCl2-pretreated and control
were sampled after 3 d-exposing to low temperature. A PicopureTM RNA
isolation Kit (Thermo Fisher Scientific, Waltham, U.S.) was used to extract the
total RNA from the leaves of CaCl2-pretreated and control of Zhennan,
and then followed by RNA purification with RNase-free DNase I (TaKaRa, Dalian,
China) and the tests of RNA degradation and contamination by using a 2% agarose
gel electrophoresis. In addition, RNA concentration, purity and integrity were
measured by using a Qubit 2.0 Fluorometer (Life Technologies, C.A., U.S.A.), a
NanoPhotometer spectrophotometer (Implen, C.A., U.S.A.) and an Agilent 2100
analyzer (Agilent Technologies, C.A., U.S.A.), respectively. A modified
procedure described by Yang and Huang (2018) was used to construct the cDNA
libraries. Transcriptome sequencing of cDNA libraries was performed on an
Illumina HiSeq 2500 platform, and 125 bp paired-end reads were generated.
Transcriptome
assembly and functional unigene annotation
The Trinity de novo assembly program was used to
assemble the remaining high-quality sequencing clean data into contigs after
filter the low-quality reads (Feng et al.
2016). The BLAST software was then used to functionally annotate all assembled
unigenes to the available sequences in public databases, including eggNOG4.5
(Huerta-Cepas et al. 2015), RefSeq
non-redundant proteins (NR) (Deng et al.
2006), euKaryotic Orthologous Groups (KOG) (Koonin et al. 2004), Swiss-Prot (Apweiler et al. 2004), Kyoto Encyclopedia of
Genes and Genomes (KEGG) (Kanehisa et al.
2004), Gene Ontology (GO) (Ashburner et
al. 2000) and Clusters of Orthologous Groups (COG) (Tatusov et al. 2000). The KEGG orthology was
obtained by using the KOBAS 2.0 program, and then then the amino acid sequence
of all unigenes were predicted. In addition, HMMER software was used to align
to Protein family (Pfam) database for obtaining the annotation information of
all unigenes (Finn et al. 2004).
Differential expression and enrichment analysis
After mapping the clean data back
onto the assembled transcriptome with TopHat software, the expression levels of
unigenes were calculated based on the fragments per kilobase of transcript per
million mapped reads (FPKM) by using Expectation maximization (RSEM) (Li and
Dewey 2011; Langmead and Salzberg 2012). And then, the differentially expressed
genes (DEGs) between the control and the CaCl2-pretreated seedlings
of Zhennan were selected by using the DESeq R package (Anders and Huber 2010).
In this study, the DEGs were characterized as the genes with an absolute log2
(fold change) value ≥ 2 and a false
discovery rate (FDR) ≦ 0.001. Based on the Wallenius’ noncentral
hypergeometric distribution, the GO enrichment analysis of DEGs was conducted
with GOseq R package (Young et al.
2010). Furthermore, the KEGG pathway enrichment analysis of the DEGs was
performed by using KOBAS software for discerning the related biochemical and
signal transduction pathways (Mao et al.
2005).
qRT-PCR validation
The sequencing data was validated
with a quantitative real-time polymerase chain reaction (qRT-PCR) analysis. In
this study, 6 candidate genes were selected, and the gene-specific primers of
these genes were designed by using the Primers Premier 5.0 program (Suppl.
Table S1). After synthesizing cDNA by using the above isolated total RNA, the
qRT-PCR was performed on a StepOnePlus Real-Time PCR Systems (Applied
Biosystems, CA, USA) with the following PCR procedure conditions: 95℃ for 120 s, 40 cycles of 95℃ for 10 s, 60℃ for 30 s, 72℃ for 30 s. The relative expression levels of the
selected genes normalized against the two Zhennan gene (pentatricopeptide
repeat-containing protein At1g43980 and DNA polymerase epsilon catalytic
subunit A-like) expression leaves and were calculated by using the 2-ΔΔCT method (Livak and Schmittgen 2001). There were 3
biological replications for each selected gene and 3 technical replications for
each biological replicate.
Statistical
analysis
S.P.S.S. version 16.0 (S.P.S.S. Inc., Chicago, I.L.,
U.S.A.) was used to perform the analysis of variance (ANOVA) and the
significant differences among the treatments were calculated with Duncan's
multiple-range test. All statistical analyses were performed at a 95%
confidence level.
Results
Effects of exogenous CaCl2 on
physiological and biochemical characteristics in cold-stressed Zhennan leaves
Eight physiological parameters were
analyzed to clarify the effects of exogenous CaCl2 on Zhennan cold
resistance. CaCl2-pretreatment significantly increased the ABA
content in Zhennan leaves (P <
0.05), while the control group possessed a significant higher ABA content after
exposed to natural low temperature (Fig. 2a). The chlorophyll content, however,
had an adverse variation pattern during the experimental period compared with
ABA in leaves (Fig. 2b). Activities of the two peroxidases
(POD and CAT) were significantly enhanced by exogenous CaCl2
before Zhennan seedlings were exposed to low temperature (0 d, P < 0.05, Fig. 2c–d). After Seedlings
were exposed to low temperature environment (3 and 15 d), the CaCl2
treatment still had a relative higher peroxidase activity, though no
statistically significant was detected. Throughout the experiment, relative
electrical and the contents of MDA, soluble sugar, and proline were
significantly increased (P < 0.05,
Fig. 2e–h). Seedlings in control group had a significant higher MDA content and
relative electrical, while CaCl2-treated seedlings had a significant
higher proline and soluble sugar contents. Furthermore, the shoot tips of control seedlings showed mild
Fig. 1: The daily mean temperature and daily minimum temperature during the
experimental periods (from December 6 to 21, 2018)
Fig. 2: Effects of exogenous CaCl2 pretreatment on abscisic acid (ABA)
(a), chlorophyll (b), peroxidase (POD) (c), catalase (CAT) (d), malondialdehyde (MDA) (e), relative electrical conductivity (f), proline (g), and soluble sugar (h)
in cold-stressed Zhennan leaves. Seedlings of Zhennan were sprayed with
distilled water (control) and 10 mmol/L CaCl2 solution for 7 d under
normal growth condition. Values within each graph followed by the different
letters indicate significant differences between treatments (lower case), and
among different sampling time (upper case) (n = 3) according to Duncan’s test (P < 0.05)
wilt, while CaCl2-treated seedling
displayed normal growth (Fig. 3). These data indicated that CaCl2
pretreatment increased the cold
tolerance of Zhennan seedlings.
Table 1: Summary of transcriptome sequencing data
Length range |
Transcript |
Unigene |
200-300 |
36,992 (18.32%) |
30,857 (30.79%) |
300-500 |
27,238 (13.49%) |
15,350 (15.32%) |
500-1000 |
45,082 (22.32%) |
19,673 (19.63%) |
1000-2000 |
51,015 (25.26%) |
18,768 (18.73%) |
≥ 2000 |
41,643 (20.62%) |
15,555 (15.52%) |
Total number |
201,970 |
100,203 |
Total length |
257,180,926 |
104,144,175 |
N50 length |
2,035 |
1,910 |
Mean length |
1273.4 |
1039.3 |
Fig. 3: Phenotypes of Zhennan seedlings under different growth conditions
throughout the 15 d low temperature treatment
Transcriptome sequencing, assembly and functional
annotation
To clarify the molecular regulation
mechanism of CaCl2 on cold resistance of Zhennan, transcriptome
sequencing was performed on a Illumina Hiseq instrument system. After filtering
out the low-quality data, approximately 40 726 813 and 54 928 919 clean reads
were obtained for the 3 d-CaCl2 and 3 d-control, respectively
(Suppl. Table S1). Data quality assessment showed that > 94.33% of these
data possessed a quality score of Q30 (i.e.,
the sequencing error rates < 0.1%), and the GC contents for CaCl2-pretreated
and control were 46.18%, and 47.48%, respectively (Suppl. Table S1).
Furthermore, 29 166 060 (71.61%) and 39 352 390 (71.64%) clean reads in two
cDNA libraries that could be successfully matched to the reference genome. The
Zhennan transcriptome was assembled by using Trinity program, the final
assembled 100 203 unigenes had a mean length of 1039.3 bp and an N50 length of
1910 bp (Table 1). These results indicated that the unigenes were qualified for
further functional annotation.
The unigenes were functionally annotated with eight public databases (GO,
Swiss-prot, NR, KOG, COG, KEGG, eggNOG and Pfam) for analyzing the gene
function information. The unigenes of Zhennan were annotated as a transcriptome
and a total of 47 781 unigenes were matched in NR database, in which 47 716
unigenes were homologous to multiple species genomes, including Nelumbo nucifera (11.99%), Macleaya cordata (7.01%), Vitis vinifera (3.76%), Quercus suber (3.43%), Hortaea werneckii (2.70%), Elaeis guineensis (2.66%), Alternaria alternate (2.36%), Phoenix dactylifera (2.22%), Baudoinia panamericana (2.04%), Aquilegia coerulea (1.94%), and other
(59.90%) (Suppl. Fig. S1).
For classifying genes and gene products, 20 712 unigenes were divided into
3 GO categories and 50 functional subcategories (Fig. 4 and Suppl. Table S2).
For the category of cellular component, the top 6 subcategories were cell (18039
unigenes, 87.1%), membrane (17149 unigenes, 82.8%), organelle (16703
unigenes, 80.6%), membrane part (15991 unigenes, 77.2%), organelle part (14476
unigenes, 69.9%), and macromolecular complex (14031 unigenes, 67.7%). For the
molecular function category, the most highly represented 4 GO terms were
catalytic activity (18436 unigenes, 89.0%), binding (18208 unigenes, 87.9%),
transporter activity (12518 unigenes, 60.4%) and structural molecule activity
(10925 unigenes, 52.7%). For biological process, the dominant groups were
metabolic process (18459 unigenes, 89.1%), cellular process (18231 unigenes,
88.0%), single-organism process (17116 unigenes, 82.6%), response to stimulus
(13656 unigenes, 65.9%), localization (14544 unigenes, 70.2%), biological
regulation (14680 unigenes, 70.9%) and cellular component organization or
biogenesis (13429 unigenes, 64.8%). Our results indicated that membrane
component-related genes were assigned to the cellular component category (GO:
0016021), while the genes related to oxidoreductase activity (GO: 0016491) and
positive regulation of abscisic acid-activated signaling pathway (GO: 0009789)
were enriched to biological process.
The COG database was also used to evaluate the homology of gene products.
A total of 14 808 unigenes were classified into 26 COG categories (Fig. 5 and
Suppl. Table S3). Carbohydrate transport and metabolism (G, 10.01%),
chaperones, posttranslational modification, protein turnover (O, 9.73%),
metabolism and lipid transport (I, 7.81%), metabolism and amino acid transport
(E, 7.64%), signal transduction mechanism (T, 7%), and cell
wall/membrane/envelope biogenesis (M, 4.03%) contained 1469, 1428, 1146, 1122,
1027, and 592 unigenes, respectively. This result indicated that these unigenes
might be involved in cold tolerance of Zhennan seedlings.
Identification and analysis of DEGs
According to the DESeq program,
4245 unigenes were characterized as the DEGs between the paired samples. Within
these DEGs, 477 unigenes were up-regulated, while 3768 unigenes were
down-regulated for the samples exposed to low temperature compared with control
group (Suppl. Fig. S2). These DEGs were functionally annotated into the 8 public
databases to further analyse the function of the DEGs: 3776 in NR, 3600 in
eggNOG, 3101 in Pfam, 2548 in GO, 2342 in KOG, 2105 in Swiss-Prot, 1680 in COG,
and 1628 in KEGG, respectively. Based on GO functional annotation, 2548 DEGs
were assigned to three categories: molecular function, cellular component and
biological process (Fig. 4). Among the category of biological process, the top
five subcategories were metabolic process (2308 DEGs), cellular process (2285
DEGs), single-organism process (2157 DEGs), localization (1946 DEGs), and
biological regulation (1894 DEGs). Within cellular component category, cell
(2305 DEGs), cell part (2304 DEGs), membrane (2301 DEGs), organelle (2199
DEGs), and membrane part (2091 DEGs) were the most highly represented. Catalytic
activity, binding, and transporter activity were the dominant subcategories in
molecular function group.
Fig. 4: Classification of all assembled unigenes and DEGs annotated in GO
database. This figure shows the ontology enrichment of DEGs in various GO
functional subcategories. All DEGs fell into the 3 categories of biological process,
celluar component and molecular function.
Fig. 5: Classification of all assembled unigenes in COG database. The horizontal
axis refers to the 26 categories, and the vertical axis indicates the number of
DEGs in each category
The
KEGG database was used for the systematic analysis of gene function. In the
present study, a total of 17 376 unigenes and 1766 DEGs were classified into
121 known pathways. The top 50 KEGG pathways were divided into four categories:
3 pathways related to cellular processes, 1 pathway connected with
environmental information processing, 13 relevant to genetic information
processing, and 33 pathways associated to metabolism (Fig. 6).
Validation of DEGs by qRT-PCR
To further verify the reliability of
the RNA-Seq data, six cold-responsive DEGs were randomly selected and analyzed
with qRT-PCR (Suppl. Table S3). The candidate genes encode
RPR (c183911), Rboh (c167842), RPM1 (190929), RPS2 (173175), HSP90 (180553) and PR1 (c192795), respectively. As
shown in Fig. 7, the log2 (fold change) values obtained by qRT-PCR
were closely consistent with the transcript abundance detected by RNA-Seq for
all of the selected unigenes, despite the difference in the absolute fold
change between the two methods. This result confirmed that the reliability of
the RNA-Seq analysis.
Discussion
Plants have evolved elaborate
mechanisms of resistance to cold stress (Ding et al. 2019). At the biochemical and physiological levels, numerous
protective substances are biosynthesized in plants, such as cold-resistance
proteins, proline, and soluble sugar (Kaplan et al. 2007). These substances are involved in
ice crystal formation, reactive oxygen species (ROS) scavenging, osmotic
potential, and regulating the stability of cell membranes (Dong et al. 2009). Of these substances, ABA
plays a very important role in frost resistance induction of plants. In the
previous studies, plants acquire increased cold tolerance upon prior drought
acclimation, a process by which the endogenous ABA in leaves increased (Mantyla
et al. 1995; Gusta et al. 1996). In addition, exogenous ABA
treatment can increase cold tolerance of plants under a non-cold acclimation
temperature condition (Gusta et al.
1996). In this study, we found a significant higher ABA content in leaves of
Zhennan before seedlings were exposed to low temperature, which indicated that
CaCl2 pretreatment has a similar effect to cold acclimation. The two
low-molecular-weight solutes, proline and soluble sugars, function as osmotic
adjustment to protect plants against the damage caused by cold stress (Ruelland
et al. 2009). While MDA and relative
electrical conductivity are usually used to reflect lipid peroxidation, and the
elevated value suggests the membrane systems may be severely damaged (Catola et al. 2016). In the present study, the
increased proline and soluble sugars, while decreased MDA and relative
electrical conductivity indicated that CaCl2-treated seedlings of
Zhennan had a higher cold resistance, which was consistent with the previous
report (Tan et al. 2011). The CaCl2
pretreatment also enhanced the activities of peroxidases (POD and CAT) before
seedlings were exposed to low temperature (Fig. 2c–d). This result agreed with
the lower MDA and relative electrical conductivity for CaCl2-treated
Zhennan.
The cold damaged plants usually showed photosynthetic inhibition, blocked
protein synthesis, and desaturation of membrane lipid (Williams et al. 1988; Shinozaki and Yamaguchi-Shinozaki 2000;
Rajkowitsch et al. 2007).
Transcriptome sequencing was performed to clarify the molecular regulation
mechanism of CaCl2 on Zhennan cold resistance. To our knowledge,
none of the literatures have dealt with the transcriptome sequencing analysis
on Zhennan. In this study, DEGs analysis revealed that abundant biological
processes associated to cold resistance have been changed by low temperature
treatment. For example, calcium-transporting ATPase activity (GO: 0005388), Ca2+
transmembrane transport (GO: 0070588), peroxidase activity (GO: 0004601),
response to oxidative stress (GO: 0006979), negative regulation of
ABA-activated signaling pathway (GO: 0009788) and
cell surface receptor signaling pathway (GO: 0007166) were markedly
down-regulated. While, calmodulin binding (GO: 0005516), protein
serine/threonine kinase activity (GO: 0004674) and protein phosphorylation (GO:
0006468) were significantly upregulated.
For organisms, biological function links closely with the coordination of
different genes. It is very important to further reveal the gene function
through analyzing the pathway of DEGs. The pathways of phagosome (18 DEGs),
peroxisome (25 DEGs), and endocytosis (43 DEGs) are associated to antioxidant
defense (Liu et al. 2018). The
phosphatidylinositol signaling system is related to signal transduction. About
78.3% DEGs were assigned to metabolism, including fatty acid metabolism, amino
acid metabolism, carbon and carbohydrate metabolism. Furthermore, several pathways are involved in
secondary metabolism, such as biosynthesis of flavonoid, terpenoid, and
alkaloid. Overall, DEGs annotated into above pathways play important role for
Zhennan seedlings resisting cold stress.
The systematic defense responses to biotic and abiotic stresses are the
important resistance mechanism for plants (Bonello et al. 2006). In the present study, the low temperature activated
the plant-pathogen interaction pathway (ko04626) in Zhennan seedlings (Fig. 8).
The previous study has found several cold-induced antifreeze proteins in leaves
of cold-acclimation plant (Thomashow 2001). The antifreeze proteins were also
found can be induced by different pathogen infection, and they are known as
pathogenesis-related protein (PR). In plants, these antifreeze proteins possess
the dual function of cold and disease resistances, so as to protect plants from
cold stress and pathogen infection. In the present study, 11 DEGs were found in
the plant-pathogen interaction pathway based on the KEGG enrichment analysis.
Among them, respiratory burst oxidase homolog protein
(Rboh, c167842), calcium-dependent protein kinase (CDPK, c173040) and disease
resistance protein RPM1 (c190929) were up-regulated, while pathogenesis-related
protein 1 (PR1, c173293, c192795), disease resistance protein RPS2 (c173175),
and molecular chaperone HtpG (HSP90, c180553) were down-regulated. In addition,
genes of calmodulin/calcium binding protein CML (CaM/CML, c164829,
c191287/c162995, c184878) showed mixed regulation.
As a second messenger, calcium ion is very important for plant to response
to environmental stimuli. The rapidly enhanced cytosolic Ca2+
concentration via Ca2+ channels after cold stress is considered as
one of the earliest cold signaling events (Anders and Huber 2010). Interestingly,
Knight et al. (1996) found that
cold-induced COR gene was Ca2+-dependent.
The more recently study indicated that G-protein regulator involved in cold
sensing through modulating calcium signals in rice (Oryza sativa) (Ma et al.
2015). These studies reveal that Ca2+ plays a key role in various
biological processes, including cold stress sensing, signal transduction, and
regulation of gene expression. In addition, numerous evidences indicate that
there is a strong linkage between Ca2+ and other messenger molecules
such as nitric oxide (NO) and ROS. For example, low ROS concentration can
promote Ca2+ influx into the cytoplasm. On the other hand, Ca2+
can regulate ROS production in plant under various stresses, such as low temperature,
heat, and drought stress (Wang et al.
2016). As such, exogenous Ca2+ application may enhance the cold
resistance of plants through activating the ion channels in the cell membrane
and further inducing
Fig. 8: The KEGG pathway of the plant-pathogen interaction. The red boxes
represent these enzymes are related to up-regulated DEGs, the green boxes refer
to the down-regulated DEGs, and the blue box indicates the enzymes were both
associated to up- and down-regulated DEGs
Fig. 7: Validation of the relative expression levels of the 6 randomly selected
DEGs by qRT-PCR. Data refer to the log2 (fold change) of expression
for each selected DEG in the cold treatment compared to control group
expression of resistance genes. Indeed, in the present study, the cold
tolerance of Zhennan seedlings were improved by exogenous Ca2+
pretreatment, and this phenomenon have been proved on the levels of phenotype
and physiological characteristics (Fig. 2 and 3). Also, the gene expression in
many metabolic pathways, including plant-pathogen interaction pathway, have
been changed by exogenous Ca2+ pretreatment.
Compared with control seedling, exogenous Ca2+ pretreatment
strongly activated the genes of PzCDPK
and PzRboh, resulting in the
accumulation of ROS by PAMP-triggered immunity and finally the defense
responses were activated by hypersensitive response (HR) and cell wall
reinforcement. The mixed regulated genes of PzCaM/CML
and disease resistance protein (PzRPM1,
PzRPS2) were not resulting in the
change of downstream genes. Interestingly, the defense-related genes induced by
WRKY transcription factor families (WRKY TFs) were not change or
down-regulated. In other plant species, WRKY TFs have been widely reported that
they can enhance the resistance of plant to various stresses and pathogen
infection (Haider et al. 2017). These
results indicated that CDPK might play key role for improving the cold
resistance of Zhennan seedlings.
Conclusion
In conclusion, our results
indicated that the exogenous CaCl2-pretreatment increased the cold
resistance of P. Zhennan seedlings,
which was verified by phenotype and physiological characteristics. The
following comparative transcriptome analysis revealed that numerous genes in
cold resistance-related biological
processes were activated, such as calcium-transporting ATPase activity, peroxidase activity, negative
regulation of ABA-activated signaling pathway, etc. In addition, the KEGG
enrichment analysis suggested that the CDPK within plant-pathogen interaction
pathway might play key role for improving the cold resistance of P. zhennan. The findings provide a novel
insight into the complexity of the Ca2+-mediated cold resistance of P. Zhennan seedlings and can improve
cold stress tolerance of this plant.
Acknowledgements
We acknowledge financial support from the National
Key Research and Development Program of China (2016YFD0600603).
Author Contributions
Bo Deng performed measurements, analyzed data and
wrote the manuscript. Botao Jia conducted the experiment, performed
measurements and analyzed data. Guihua Liu conceived the experimental idea and
provided field support in establishing the experiment. Xiaoyan Zhang
established the experiment and performed measurements.
References
Anders S, W Huber (2010). Differential expression analysis for sequence
count data. Genome Biol 11; Article R106
Apweiler R, A Bairoch, CH Wu, WC Barker, B Boeckmann, S
Ferro, E Gasteiger, HZ Huang, R Lopez, M Magrane, MJ Martin, DA Natale, CO
Donovan, N Redaschi, LL Yeh (2004). UniProt: The universal protein knowledge
base. Nucl Acids Res 32:115‒119
Ashburner M, CA Ball, JA Blake, D Botstein, H Butler, JM
Cherry, AP Davis, K Dolinski, SS Dwight, JT Eppig, MA Harris, DP Hill, L
Issel-Tarver, A Kasarakis, S Lewis, JC Matese, JE Richardson, M Ringwald, GM
Rubin, G Sherlock (2000). Gene ontology: Tool for the unification of biology. Nat Genet 25:25‒29
Bates LS, RP Waldren, ID Teare
(1973). Rapid determination of free proline for water-stress studies. Plant Soil 39:205‒207
Bonello P, TR Gordon, DA Herms, DL
Wood, N Erbilgin (2006). Nature and ecological implications of pathogen-induced
systemic resistance in conifers: A novel hypothesis. Physiol Mol Plant Pathol 68:95‒104
Buysse J, R Merckx (1993). An
improved colorimetric method to quantify sugar content of plant tissue. J Exp Bot 44:1627‒1629
Cao J, H Shang, Z Chen, Y Tian, H
Yu (2016). Effects of elevated ozone on stoichiometry and nutrient pools of Phoebe bournei (Hemsl.) Yang and Phoebe zhennan SLee et FNWei seedlings
in subtropical China. Forests 7;
Article 78
Catola S, G Marino, G Emiliani, T Huseynova, M Musayev, Z Akparov, BE
Maserti (2016). Physiological and metabolomic
analysis of Punica granatum (L.)
under drought stress. Planta 243:441‒449
Chen Z, J Cao, H Yu, H Shang
(2018). Effects of elevated ozone levels on photosynthesis biomass and
non-structural carbohydrates of Phoebe
bournei and Phoebe zhennan in
subtropical China. Front Plant Sci 9;
Article 1764
Cui J, N Jiang, XX Zhou, XX Hou, GL Yang, J Meng, YS Luan
(2018). Tomato MYB49 enhances resistance to Phytophthora
infestans and tolerance to water deficit and salt stress. Planta 248:1487‒1503
Deng YY, JQ Li, SF Wu, YP Zhu, YW Chen, FC He (2006).
Integrated nr database in protein annotation system and its localization. Comput Eng 32:71‒74
Ding YL, YT Shi, SH Yang (2019). Advance and challenges
in uncovering cold tolerance regulatory mechanisms in plants. New Phytol 222:1690-1704
Dong CH, BK Zolman, B Bartel, BH Lee, B Stevenson, M Agarwal, JK Zhu
(2009). Disruption of Arabidopsis CHY1 reveals an important role of metabolic status in
plant cold stress signaling. Mol Plant
2:59‒72
Draper HH, M Hadley (1990).
Malondialdehyde determination as index of lipid-peroxidation. Meth Enzymol 186:421‒431
Feng YZ, L Zhang, JM Fu, FD Li, L
Wang, XF Tan, WJ Mo, HP Cao (2016). Characterization of glycolytic pathway
genes using RNA-Seq in developing kernels of Eucommia ulmoides. J Agric Food Chem 64:3712‒3731
Finn RD, A Bateman, J Clements, P
Coqqill, RY Eberhardt, SR Eddy, A Heger, K Hetherington, L Holm, J Mistry, EL
Sonnhammer, J Tate, M Punta (2004). Pfam: The protein families database. Nucl Acids Res 42:222‒230
Fu JJ, Y Wu, YJ Miao, YM Xu, EH
Zhao, J Wang, HE Sun, Q Liu, YW Xue, YF Xu, TM Hu (2017). Improved cold tolerance
in Elymus nutans by exogenous
application of melatonin may involve ABA-dependent and ABA-independent pathways.
Sci Rep 7; Article 39865
Gao J, W Zhang, J Li, H Long, W He,
X Li (2016). Amplified fragment length polymorphism analysis of the population
structure and genetic diversity of Phoebe
zhennan (Lauraceae) a native species to China. Biochem Syst Ecol 64:149‒155
Gusta LV, RW Wilen, P Fu (1996). Low-temperature stress tolerance: The role of abscisic acid sugars and
heat-stable proteins. Hortic Sci 31:39‒46
Haider MS, MM Kurjogi, M
Khalil-Ur-Rehman, M Fiaz, T Pervaiz, ST Jiu, HF Jia, W Chen, JG Fang (2017).
Grapevine immune signaling network in response to drought stress as revealed by
transcriptomic analysis. Plant Physiol
Biochem 121:187‒195
Hu Y, B Wang, TX Hu, H Chen, H Li,
W Zhang, Y Zhong, HL Hu (2015). Combined action of an antioxidant defence
system and osmolytes on drought tolerance and post-drought recovery of Phoebe zhennan SLee saplings. Acta Physiol Plantarum 37; Article 84
Huang YT, WJ Qian, B Wang, HL Cao,
L Wang, XY Hao, XC Wang, YJ Yang (2015). Effects of exogenous calcium and
inhibitors of calcium signaling transduction pathway on cold resistance of tea
plant. J Tea Sci 35:520‒526
Huerta-Cepas J, D Szklarczyk, K Forslund, H Cook, D
Heller, MC Walter, T Rattei, DR Mende, S Sunagawa, MKLJ Jensen, CV Mering, P
Bork (2015). eggNOG 4.5: A hierarchical orthology framework with improved
functional annotations for eukaryotic prokaryotic and viral sequences. Nucl Acids Res 44:286‒293
Janmohammadi M, L Zolla, S
Rinalducci (2015). Low temperature tolerance in plants: Changes at the protein
level. Phytochemistry 117:76‒89
Kanehisa M, S Goto, S Kawashima, Y Okuno, M Hattori
(2004). The KEGG resource for deciphering the genome. Nucl Acids Res 32:277‒280
Kaplan F, J Kopka, DY Sung, W Zhao, M Popp, R Porat, CL Guy (2007). Transcript and metabolite profiling during cold acclimation of Arabidopsis reveals an intricate
relationship of cold-regulated gene expression with modifications in metabolite
content. Plant J 50:967‒981
Knight H, AJ Trewavas, MR Knight
(1996). Cold calcium signaling in Arabidopsis
involves two cellular pools and a change in calcium signature after
acclimation. Plant Cell 8:489‒503
Koonin EV, ND Fedorova, JD Jackson, AR Jacobs, DM Krylov,
KS Makarova, R Mazumder, SL Mekhedov, AN Nikolskaya, BS Rao, LB Rogozin, S
Smirnov, AV Sorokin, AV Sverdlov, S Vasudevan, YI Wolf, JJ Yin, DA Natale
(2004). A comprehensive evolutionary classification of proteins encoded in
complete eukaryotic genomes. Genome Biol
5:1-28
Langmead B, SL Salzberg (2012).
Fast gapped-read alignment with Bowtie 2.
Nat Meth 9:357‒359
Leites LP, GE Rehfeldt, KC Steiner (2019). Adaptation to
climate in five eastern North America broadleaf deciduous species: Growth
clines and evidence of the growth-cold tolerance trade-off. Perspect Plant Ecol 37:64‒72
Li B, CN Dewey (2011). RSEM: Accurate transcript
quantification from RNA Seq data with or without a reference genome. BMC Bioinform 12; Article 323
Liu Y, ZZ Xin, J Song, XY Zhu, QN
Liu, DZ Zhang, BP Tang, CL Zhou, LS Dai (2018). Transcriptome analysis reveals
potential antioxidant defense mechanisms in Antheraea
pernyi in response to zinc stress. J
Agric Food Chem 66:8132‒8141
Livak KJ, TD Schmittgen (2001). Analysis of relative gene expression data using real-time quantitative PCR
and the 2−ΔΔCT method. Methods 25:402‒408
Ma Y, X Dai, Y Xu, W Luo, X Zheng,
D Zeng, Y Pan, X Lin, H Liu, D Zhang, J Xiao, X Guo, S Xu, Y Niu, J Jin, H
Zhang, X Xu, L Li, W Wang, Q Qian, S Ge, K Chong (2015). COLD1 confers chilling
tolerance in Rice. Cell 160:1209‒1221
Mantyla E, V Lang, ET Palva (1995). Role of abscisic acid
in drought-induced freezing tolerance cold acclimation and accumulation of
LTI78 and RAB18 proteins in Arabidopsis
thaliana. Plant Physiol 107:141‒148
Mao X, T Cai, JG Olyarchuk, L Wei (2005). Automated genome annotation and
pathway identification using the KEGG orthology (KO) as a controlled
vocabulary. Bioinformatics 21:3787‒3793
Paul MJ, SP Driscoll (1997). Sugar
repression of photosynthesis: The role of carbohydrates in signaling nitrogen
deficiency through source: Sink imbalance. Plant
Cell Environ 20:110‒116
Perochon A, D Aldon, JP Galaud, B
Ranty (2011). Calmodulin and calmodulin-like proteins in plant calcium
signaling. Biochimie 93:2048‒2053
Rajkowitsch L, D Chen, S Stampfl, K
Semrad, C Waldsich, O Mayer, MF Jantsch, R Konrat, U Blasi, RRN Schroeder
(2007). A chaperones RNA annealers and RNA helicases. RNA Biol 4:118‒130
Rooy SSB, GH Salekdeh, M Ghabooli, M Gholami, R Karimi
(2017). Cold-induced physiological and biochemical responses of three grapevine
cultivars differing in cold tolerance. Acta
Physiol Plantarum 39:264-276
Ruelland E, MN Vaultier, A Zachowski, V Hurry (2009). Cold signalling and cold acclimation in plants. In: Advances in Botany Research, Vol. 49, pp: 35‒150. Kader JC, M
Delseny (Eds.). Elsevier Ltd.
Shinozaki K, K Yamaguchi-Shinozaki
(2000). Molecular responses to dehydration and low temperature: Differences and
cross-talk between two stress signaling pathways. Curr Opin Plant Biol 3:217‒223
Tan W, QW Meng, M Brestic, K Olsovska, XH Yang (2011). Photosynthesis is improved by
exogenous calcium in heat-stressed tobacco plants. J Plant Physiol 168:2063‒2071
Tatusov RL, MY Galperin, DA Natale (2000). The COG
database: A tool for genome scale analysis of protein functions and evolution. Nucl Acids Res 28:33‒36
Thomashow M (2001). So, what’s new
in the field of plant cold acclimation? Lots! Plant Physiol 125:89‒93
Trapnell C, BA Williams, G Pertea,
A Mortazavi, G Kwan, MJV Baren, SL Salzberg, H Upadhyaya, SK Panda, BK Dutta
(2011). CaCl2 improves post-drought recovery potential in Camellia sinensis (L.) OKuntze. Plant Cell Rep 30:495‒503
Wang WH, EM He, Y Guo, QX Tong, HL
Zheng (2016). Chloroplast calcium and ROS signaling networks potentially
facilitate the primed state for stomatal closure under multiple stresses. Environ Exp Bot 122:85‒93
Williams JP, MU Khan, K Mitchell, G Johnson (1988). The effect of temperature on the level and biosynthesis of unsaturated
fatty acid in diacylglycerols of Brassica
napus leaves. Plant Physiol 87:904‒910
Xie JL, JQ Qi, XY Huang, N Zhou, Y
Hu (2015). Comparative analysis of modern and ancient buried Phoebe zhennan wood: Surface color
chemical components infrared spectroscopy and essential oil composition. J For Res 26:501‒507
Yang TY, XS Huang (2018). Deep
sequencing-based characterization of transcriptome of Pyrus ussuriensis in responses to cold stress. Gene 661:109‒118
Young MD, MJ Wakefield, GK Smyth, A Oshlack (2010). Gene ontology analysis for
RNA-Seq: Accounting for selection bias. Genome
Biol 11:14-25