Introduction
Rehmannia glutinosa L. (Scrophulariaceae)
is one of the commonly used Chinese herbal medicines including many important
medicinal active components, such as catalpol, reotide, motherwort glycosides,
rhubarb polysaccharides, amino acids, stigmasterol, and relevant
pharmacological compounds (Zhang et al.
2020; Chen et al. 2021). Modern
pharmacological studies shown that R. glutinosa
owned antioxidant, neuroprotective, anti-aging, and other pharmacological effects
(Li and Meng 2015; Wang et al. 2015).
However, replanting disease seriously limited the production in the cultivation
of R. glutinosa. Replanted R. glutinosa grown slowly and suffered
from pests and diseases, the leaf area and chlorophyll content of replanted R. glutinosa reduced obviously, resulting
in the roots with inability formatted into tuberous roots, which seriously
decreased the yield, quality, and active ingredient accumulation (Li et al. 2012a, b; Guo et al. 2013; Yang et al. 2015; Wang et al.
2021). The replanting disease has attracted thus wide
attention that required urgent solving in production of R. glutinosa.
Numerous studies shown that replanting disease caused by the changes of
the soil properties, the allelochemical autotoxins, and the imbalance
of the rhizosphere microbials (Zhu et al.
2007; Zhang et al.
2010). In previous study, many allelochemical autotoxins derived from R. glutinosa including some phenolic
acids and flavonoids (Chi et al. 2013). Furthermore, the rhizosphere microbes around replanted R. glutinosa were found to transform
from bacteria to fungi, leading to continuous proliferation of numerous
pathogenic microorganisms (Lin et al.
2011). In addition, the imbalance of the rhizosphere microbials has been
confirmed to mainly induced by rhizosphere allelochemical autotoxins (Wu et al. 2015; Zhang et al. 2018). Based on transcriptomics approach, previous studies found
that many important signaling pathway including immune responses, ROS
generation, programmed cell death, lignin synthesis, Ca2+ signal
transduction and ethylene synthesis were significantly activated in replanted R. glutinosa (Yang et al. 2015; Li et al.
2017). It remained unclear that how these signals were produced under
replanting stress and further resulted in the death of replanted plants.
In the process of plants response to environmental factors, the
cellular membrane system plays an important role in sensing of environmental changes (Mansour 2012). The plant membrane
system was an important subcellular structure that divided different cell units
each other, including the plasma membrane, tonoplast, mitochondrial membrane etc. The plant membrane system has been
confirmed to play an essential role in responding and sensing external stress
factors, such as low temperature, salt, and drought (Osakabe et
al. 2013; Janicka-Russak and Kabala 2015). Replanting disease
also constitutes a particular type of stress that is associated with
allelochemical autotoxins, harmful microorganisms, and their interaction. As
the continuous proliferation of rhizosphere microbes and accumulation of
allelochemicals, there were many specific receptors in plasma membrane might be
used to respond to the stress in replanted R. glutinosa. These receptors likely
further induced downstream response. In addition to the
receptors presented in the plasma membrane, many channels
and receptors located in intracellular membrane were
found closely relate to stress response and cell death
(Huang et al. 2018). Numerous studies have proved that the formation of replant disease
was induced by the micro-ecological imbalance in rhizosphere mediated by allelochemical
autotoxins (Qiu et al. 2010; Li et al. 2012a, b; Zhao et al. 2016). However, the replant soil
in the rhizosphere was only harmful to R.
glutinosa but has no harmful effect on other crops. This indicated that there was a specific signal
transduction system in R. glutinosa
to sense and respond to the replant stress. Membrane receptor protein is a key
factor for plants to respond to environmental stress, replant disease is a
complex stress for R. glutinosa.
Previous studies have found that the root vitality of replanted R. glutinosa was very low and the
membrane peroxidation was seriously (Wu et
al. 2015). However, the studies did not involve in membrane proteins that
specifically respond to replant stress, and how the plants receive, transmit
and respond to the signals was also unknown. The reception of signals is the
prerequisite of signal transduction; receptor proteins located in the membrane were
the main channels for plants to perceive signals from the external environment.
Therefore, study on the membrane receptor proteins in replanted R. glutinosa has
the great significance to clarify the death behavior-inducing by replanted
practice.
The successful extraction of membrane proteins was important basis for
the functional study of differential membrane proteins. The membrane protein
extraction methods in animals are relatively established, while the opposite is
true for plants. Two main ways of membrane proteins extraction currently exist:
one is the two-phase partition method, and another is the membrane protein
extraction kit (Pang et al. 2010; Pan et al.
2013). In this study, the extraction efficiency of two ways were
compared to obtain a suitable method for the membrane proteins
extraction of tuberous roots. Based on above optimized methods, membrane
proteins were extracted from the tuberous roots of R. glutinosa. Simultaneously, to screen critical membrane receptor
proteins in response to replanting stress, the abundance
level and kinds of membrane proteins in R.
glutinosa samples identified through iTRAQ technologies. The expression pattern
of receptor proteins which under the factors of microbes and allelochemical
autotoxins that closely related to replanting disease formation were
investigated by qRT-PCR methods. This study provided a
theoretical basis for revealing the molecular mechanisms of replanting stress
in R. glutinosa.
Materials and Methods
Experimental materials
In this study, the R. glutinosa (cultivar "Wen 85-5",
which was widely planted and had high medicinal value, and replanting disease
severely affected its yield and medicinal quality) was cultivated in the
experimental field which located in Jiaozuo City, Henan province, China (34º48'
N to 35º30' N, 112º02' E to 113º 38' E), an R.
glutinosa production area. The R. glutinosa
were named as the first planted R. glutinosa
(FP) which were planted in a section of the experimental field where R. glutinosa had not been planted for 10
years. Moreover, R. glutinosa were
named as the second planted R. glutinosa (SP)
which were planted in the experimental field where R. glutinosa had been planted in the previous year. A previous
study showed that the damage of SP R. glutinosa
was most serious at about 90 days after planting (Yang et al.
2014). During the key period of replanting damage to R. glutinosa, we obtained the root
samples of FP and SP, three biological replicates respectively, each replicate
was 3‒7 plants, we collected root tissues (the entire roots of the FP and SP,
pooled and mixed respectively). The samples were collected and stored at -80°C in preparation for downstream experiments.
Protein extraction and extraction efficiency analysis of the R. glutinosa root samples
In order to select the appropriate membrane protein extraction method
of R. glutinosa root tissues, the membrane
protein kits and two-phase partition method were used to analyze the
concentrations and quality of the extracted membrane proteins. Then the most
efficient method was used to extract the membrane proteins of FP and SP samples
(three biological replicates respectively, each replicate was 3‒7 plants).
Method 1: Membrane protein extraction kit A (MinuteTM
total membrane and plasma membrane extraction kit SM-005; Invent
Biotechnologies, Plymouth, Minnesota, USA) (Zhang et al. 2019; Su et al.
2021; Yuan et al. 2021). Extraction processes:
1–1.5 g R. glutinosa root tissue
samples were grinded into powder in liquid nitrogen and placed into 10‒15 mL centrifuge
tubes to which 2 mL of buffer A was added. The tubes were mixed for 2‒3 min, centrifuged
for 5 min at 500 × g and 4°C, and then
transferred the supernatant to a new 2 mL centrifuge tube. The tube was
centrifuged for 10 min at 10,000 × g and 4°C, and transferred the supernatant to a new 2.0 mL centrifuge tube and
centrifuged for 30 min at 14,000 × g and 4°C. After removing the supernatant (cytoplasmic protein), the remaining
pellets was resuspended using 0.3 mL of buffer B by a pipette gun. The
resuspension was then transferred to a new centrifuge column and centrifuged
for 10 min at 14,000 × g and 4°C, transferred
the supernatant to a new 2 mL centrifuge tube to which 1.6 mL of pre-cooled PBS
was added. The tube was inverted several times to mix the solution and then
centrifuged for 30 min at 14,000 × g and 4°C. The resulting precipitate (pellet) was membrane proteins.
Method 2: Membrane protein extraction kit B (BB-3152-2
plant membrane protein extraction kit; Best Bio Biology) (He et al. 2018; Zhuang et al. 2019; Du et al.
2020). Extraction processes: 2 μL
of protease inhibitor was placed into each 500 μL extract A as a reference, and the extract premix was
prepared, mixed well, and set it on ice. Then, 1‒1.5 g of R. glutinosa root tissue samples were grinded into powder in liquid
nitrogen and added to a pre-cooled 10‒15 mL centrifuge tube to which 2
mL extract A was added. The tube was mixed and oscillated at 2‒8°C for 2–3 h and then centrifuged for 5 min at 12,000 × g and 2–8°C. Following this, 40 μL
extract B was added to the supernatant and mixed thoroughly, and then placed in
water bath at 37°C for 10 min. The solution was
then centrifuged for 5 min at 1,000 × g and 37°C. Then the solution had divided into two layers, and the upper layer was
removed carefully, leaving about 200 μL
of liquid at the bottom of the tube. The solution was diluted with 1 to 2
volumes of the membrane protein solution to obtain the membrane protein
samples.
Method 3: Two-phase partition method (Santoni 2007). For
this method, 1‒1.5 g of root samples were grinded into powder
in liquid nitrogen and placed into 10‒15 mL pre-cooled centrifuge
tubes to which 10 mL pre-cooled protein extract was added. The solutions were pumping
evenly with a pipette, centrifuged for 15 min at 8,000 × g and 4°C, and transferred the supernatant to a new centrifuge
tube, centrifuged for 1 h at 100,000 × g and 4°C (supernatant is the cytoplasmic protein, the pellet is a microsomal
fraction). Then, the supernatant was removed and the extraction solution was
re-added to the pellet and centrifuged for 1 h at 100,000 × g and 4°C, then, removed the supernatant and the pellet dissolved
in a certain amount of extraction solution, and then stored at -80°C.
Extraction efficiency analysis of the different membrane protein
extraction methods
The protein integrity was detected by
SDS-PAGE (Sodium Dodecyl Sulfate Polyactylamide Gel Electrophoresis). The
extraction efficiency of R. glutinosa root tissue membrane proteins
was quantitatively analyzed by BCA kit (Easy II Protein Quantitative
Kit, Trans Gen Biotech). The specific analysis processes were as follows: bovine serum albumin (BSA) standard solution was diluted to 500 μg/mL with PBS (Phosphate Buffer Saline).
Based on the number of samples, the BCA (Bicinchoninic
acid) Solution A and BCA Solution B were diluted in a 50:1 ratio into the
working solution, mixed well, and set aside for 24 h at 4°C. Then, eight 1.5 mL centrifuge tubes were numbered respectively
and used for the compound of diluted standard solution. After diluting the
sample to be tested 10 times with 1 × PBS, 50 μL of the solution was added to 500 μL of BCA working solution. The tube was mixed by oscillation
and then placed at 37°C for 30‒90 min. 5 μL of the reaction product was then obtained and placed in
centrifuge tube A as a negative control. The absorbance of the sample was
measured with a NanoDrop 2000 spectrophotometer (http://thermofisher.biomart.cn) at a wavelength of 562 nm and a
standard curve was constructed to calculate the concentration of the protein
samples. Then, 5×loading buffer was added to 20 μg of each diluted protein sample, and then placed in a
boiling water bath for 5 min. These were assessed by 12.5% SDS-PAGE
electrophoresis (constant current 14 mA, 90 min), stained with Coomassie
brilliant blue, and the BSA protein quantified by a spectrophotometer.
Classification and identification of the proteins obtained by different
membrane protein extraction methods
Protein samples with
higher extraction efficiency were subjected to protein reductive alkylation and
enzymolysis. Based on the FASP (Filter Aided Proteome
Preparation) method (Wisniewski et al. 2009) for the enzymolysis of protein, the specific processes were as follows: 20 μg
quantitative protein sample was added to five volumes of pre-cooled acetone and
set aside at -20°C for 1 h, after which the
protein had fully precipitated. Then, centrifuged for 10 min at 12,000 × g and
4°C and the precipitate (pellet) were freeze-dried by vacuum
concentration. 10 μL of the
protein re-dissolving solution was used to fully dissolve the protein
precipitate, to which 40 μL of
protein reduction solution was added and then reacted at 37°C for 1 h. Following this, 40 μL of protein alkylation solution (9 mol/L urea, 50
mmol/L IAA, 50 mmol/L NH4HCO3) was added and the solution
was then reacted at room temperature for 10 min. The reduced alkylated protein
solution was then added to a 10 kD ultrafiltration tube, centrifuged for 20 min
at 12,000×g, and then discarded the bottom solution in the collection tube.
Ammonium bicarbonate (150 μL 1
mmol/L) was added and centrifuged for 20 min at 12,000 × g. Once again, the
bottom solution of the collecting tube was discarded, and this process was
repeated three times. The resulting solution was placed in a new collection
tube to which 100 μL sequencing
grade tryps in solution (5 ng/μL)
was added, and the reaction continued at 37°C for 12 h. Following this, the tube was centrifuged for 10 min at
12,000 × g and the digested peptides were collected. 50 μL of ammonium bicarbonate (1 mmol/L) was added to the ultrafiltration
tube and centrifuged for 10 min at 12,000 × g. The bottom solution was
collected from the tube and combined with the previous solution, and then
freeze-dried (lyophilized). To identify the properties of the proteins, the
peptide samples obtained after proteolysis were detected by mass spectrometry.
Extraction and purification of the root tissue membrane proteins of FP
and SP R. glutinosa
We used the method 2 (membrane protein extraction kit B) to extract the
membrane proteins of R. glutinosa
root tissue samples, using the resulting samples for the downstream
experiments. Specifically, 30-fold volume (m/v) of SDS (sodium dodecyl sulfate)
lysis buffer was added to the resulting membrane protein sample and the pellet
was re-suspended by vortex mixing and then placed in a boiling water bath for 5
min, sonicated (80 W, 10 s on, 15 s off, 10 cycles), and then placed back in
the boiling water bath for 15min. Following centrifuged for 40 min at 14,000×g,
filtered the supernatant through the 0.22 μm
membrane and collected the filtrate. The BCA method was used for protein
quantification: the samples were aliquot and stored at -80°C. Taken 20
µg of protein from
each sample, to which 5 × loading buffer was added, and the tubes were placed
in a boiling water bath for 5 min. The protein was quantified using 12.5%
SDS-PAGE electrophoresis (constant current 14 mA, 90 min) and Coomassie
Brilliant Blue staining. And isobaric tag for relative and absolute
quantitation (iTRAQ) analysis was used for the extracted protein samples, and the
samples were also chromatographed and analyzed by Q-Exactive mass spectrometry.
Data identification method
The
original mass spectrometry analysis data were in RAW file format, Mascot 2.2 (Matrix
Science) and Proteome Discoverer 1.4 software (Thermo Fisher Scientific) were used
for library identification and quantitative analysis (Huang et al. 2013). The Gene Ontology (GO) function annotation of the target protein collection using Blast 2 GO, the process can be
roughly divided into four steps: Blast, GO entry extraction, GO annotation, and
supplementary annotation. In the Kyoto Encyclopedia of Genes and Genomes (KEGG)
database, KO (KEGG Orthology) is a classification system of genes and their
products. Orthologs and their products with similar functions in the same
pathway are grouped together and given the same KO (or K) tag. The Pathway
Enrichment Analytics free online database analysis software tool Omic Share
(www.omicshare.com/tools) was used for the
enrichment analysis.
PCR detection of pathogen infection in R. glutinosa
Fusarium oxysporum f. spp. is a specific pathogen
that our research team isolated from the rhizospheric soil of replanted R. glutinosa. Our preliminary
experiments have confirmed that the rhizosphere allelochemical autotoxin of SP R. glutinosa can significantly promote F. oxysporum proliferation (Li et al.
2016). In this study, we used F. oxysporum to infect R. glutinosa,
and obtained root samples when the infected R.
glutinosa started exhibiting symptoms of infection. The root samples were
subjected to electron microscopic analysis and DNA extraction. PCR primers were designed for the pathogenic fungi
according to the reported conserved sequences in the fungal ribosomal
transcribed spacer region. The primers of F.
oxysporum are ITS1-F (5'- CTTGGTCATTTAGAGGAAGTAA-3') and ITS4-R (5'-TCCTCCGCTTATTGATATGC-3')
were synthesized with reference to Wu et
al. (2015) (Sun Ya Biotechnology Co., Ltd. Fuzhou, China). The PCR reaction
mixture was 20 μL, consisting of
1.6 μL of dNTPs (25 μmol/L), 2.0 μL of 10 × ETaq buffer (containing Mg2+), 1 μL of primer (10 μmol/L), 0.1 μL of ETaq enzyme, 1 μL
template DNA (10 ng), and 13.3 μL
H2O (Sangon Biotech). The PCR reaction conditions were as follows:
pre-denaturation at 95°C for 5 min,
followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 45 s,
extension at 72°C for 1 min, and a final
extension at 72°C for 3 min. After the reaction,
the products were stored at 4°C for later
use. DL Marker 2000 (Sangon Biotech) was used as a standard molecular marker
and the PCR products were detected by 1% agarose gel electrophoresis. The relative gene expression of the genes was calculated using the 2-△△Ct method (Livak and Schmittgen 2001).
Results
Validity analysis of the membrane proteins extraction methods
In order to effectively extract membrane
proteins from the root tissue of R. glutinosa, three methods were used for extraction respectively. As
a result, there were obvious differences in the protein concentrations
obtained by the three methods, in which, 23 μg/mL
of the protein abundance extracted by method 1, 400 μg/mL of the protein abundance extracted by method 2, and 580 μg/mL of the protein abundance
extracted by method 3. The protein concentration obtained by method 1 was
relatively lower in comparison to other two methods and did not fully meet the
requirements for further protein experiments. Thus, method 2 and method 3 were
further compared for the efficiency of membrane protein extraction. The
candidate membrane proteins obtained by method 2 and method 3 were analyzed by
LC-MS/MS, and the peptide fragments obtained were aligned with the database of R. glutinosa total proteins. There were 870
and 526 proteins were extracted by the method 2 and method 3, respectively, of
which, 532 and 289 proteins were respectively clearly annotated as membrane
proteins. Therefore, method 2 indicated have better enrichment efficiency of
membrane proteins compared to the method 3. Finally, method 2 was used for the
differential membrane proteins analyses of the FP and SP R. glutinosa.
Membrane protein library construction of the FP and SP R. glutinosa
To study the differentially expressed membrane proteins between the FP
and SP R. glutinosa, the membrane
proteins of the FP and SP R. glutinosa root
tissues (each sample three biological replicates) were extracted by method 2. The acquired proteins were identified by the iTRAQ technique. By
comparing the results of iTRAQ and LC-MS/MS with earlier constructed R. glutinosa proteomics database, a
total of 4,285 proteins were obtained from the FP and SP, including 1,753
non-annotated and 2,532 annotated proteins in the UniProtKB database. Moreover,
1,465 out of 2,532 were identified as membrane proteins, which contain plasma
membrane, chloroplast membrane, mitochondrial membrane, Golgi membrane etc. The
extraction rate of membrane proteins reached 57.86% (Fig. 1c). The highest
peptide coverage was 73.13% and there were 1,700 proteins with a coverage
degree over 10. A total of 1,837 proteins were detected in the samples of SP R. glutinosa, of which 633 had ≥ 2
unique peptides. A total of 2,541 proteins were detected in the FP R. glutinosa, of which 1,006 had ≥
2 unique peptides. The obtained protein sequences provided the basis for the
further identification of differential membrane proteins between the FP and SP R. glutinosa.
Difference analysis of the root membrane proteins
between the FP and SP R. glutinosa
In order to identify the membrane proteins which
respond to replanting stress, we screened out 912 differential proteins between
the FP and SP samples based on the P
< 0.05 and > 1.2 fold. A total of 214 non-membrane proteins were removed
from the 912 differentially expressed proteins through the UniProt online
software. The remaining 698 proteins were annotated as membrane proteins, of
which, there were 339 membrane proteins were up-regulated, 359 membrane proteins
were down-regulated in the SP R. glutinosa
(Fig. 1a, b).
To analyze the functions of the differential membrane
proteins between the FP and SP R. glutinosa,
we assessed those proteins using GO and KEGG. Among up-regulated expressed
membrane proteins in the SP R. glutinosa,
a total of 339 differential membrane proteins matched to 5,341 GO entries, 330
GO terms were obtained by GO enrichment analysis. Those proteins mainly involved in trans-membrane transport,
vesicle-mediated transport, intracellular transport, protein transport, and ion
transport (Fig. 2a, Fig. 3a). Among the 359 down-regulated membrane proteins in
the SP R. glutinosa, a total of 336
GO terms were obtained by GO enrichment analysis, mainly included response to
chemical stimulation, metabolism of small-molecule metabolites, and response to
stress (Fig. 2b, Fig. 3b). Cellular location analysis showed that the
differential membrane proteins were localized to the cellular membrane (GO:
0016020 [1.64e-13]).
KEGG functional analysis was performed on the screened
differentially expressed membrane proteins. There were 340 metabolic pathways
were significantly enriched, of which, 161 pathways in carbohydrate metabolism (such
as starch and sucrose metabolism, glycolytic or gluconeogenesis, pentose
phosphate pathway, and ascorbate metabolism), 18 pathways in plant-pathogen
interaction, 42 pathways were participated in protein folding, sorting, and
degradation processes (endoplasmic reticulum protein synthesis, protein export,
RNA degradation etc.), 56 in transcriptional
translation processes (such as ribosomes, RNA transport), 47 in protein
transport and degradation pathways, including endocytosis, peroxisomes,
phagosomes and autophagy, and 24 in signal transduction pathways and membrane
trafficking (e.g., plant hormone
signaling, phosphoinositide signaling transduction, ABC transporter). In
addition, 30 significantly enriched pathways were associated with metabolite
transport pathways, including endoplasmic reticulum protein processing, amino
acid biological processes, carbon metabolism, sucrose and starch metabolism,
oxidative phosphorylation, and secondary metabolism (terpenoids, polyketides
metabolism etc.) (Fig. 4a). The up-regulated
expressed membrane proteins in SP R. glutinosa
were mainly involved in protein synthesis in the endoplasmic reticulum,
glycerophospholipid metabolism, oxidative phosphorylation, endocytosis,
phagosomes, sucrose and starch metabolism, protein export, quinone
biosynthesis, and other secondary metabolism (Fig. 4b).
The down-regulated expressed membrane proteins in SP R. glutinosa were primarily involved in carbon metabolism, amino
acid biosynthesis, glycolysis and gluconeogenesis, peroxisomes, pentose
phosphate pathway, and carbon assimilation within photosynthetic tissues (Fig.
4c).
Integration analysis of differentially expressed membrane proteins between
FP and SP R. glutinosa root samples
%20337-348_files/image002.png)
Fig. 1: Differently expressed proteins in root tissue of FP and SP R. glutinosa (a) iTRAQ
protein differential expression profile: P-value
< 0.05, the threshold of variation difference was 1.2 folds, which means
that SP/FP values more than 1.2 folds or less than 0.83 were considered
differentially expressed, (b)
Analysis and statistics of the differentially expressed proteins, (c) Positional distribution of differential proteins in root tissue of SP and FP R. glutinosa
FP= the R. glutinosa
first planted in the field; SP= the R. glutinosa replanted in the same field continuously
%20337-348_files/image004.jpg)
Fig. 2: GO enrichment analysis of the biological process part,
cellular component part, and molecular function part about differently
expressed membrane proteins (a)
significantly up-regulated expressed membrane proteins in replanted R. glutinosa, (b) significantly down-regulated
expressed membrane proteins in replanted R.
glutinosa
In order to describe the function of differential membrane proteins in more
detail, a comprehensive functional classification of those proteins was
conducted based on the annotations of the GO, KEGG, and Nr databases. The
results indicated that differential membrane proteins were
involved in various metabolic pathways, including transport and breakdown, signal
transduction, terpene and polyketide metabolism, membrane trafficking and
environmental response (Table 1). We also identified a large number of membrane
receptor proteins (such as LRR-RLK,
G-protein coupled receptors, interleukins) in the SP R. glutinosa. It is noteworthy that there were 18 differential
membrane proteins involved in plant pathogen interaction, including LRR-RLK protein kinase, calmodulin, and
calcium-dependent protein kinase, which indicated that the pathogenic bacterial
invasion is one of the factors inducing replanting disease of R. glutinosa. In addition, 44 proteins
involved in endocytosis, phagosomes, and peroxisomes, indicated that metabolism
and signal transduction in SP R. glutinosa
were active. At the same time, there were 13 proteins involved in the sensing
and transduction of hormonal signals (including gibberellin, auxin, and
brassinolide), which implied that hormones played the important regulatory role
in response to replanting stress.
Table 1: The main classifications of the differentially expressed
membrane proteins between FP and SP R. glutinosa
|
Class |
Pathway |
Protein Number |
Proteins |
|
Transport and catabolism |
Endocytosis |
22 |
PRAF1、PIP5K、DNM、HSPA4、RAB7A、VPS23、VPS28、VPS4… |
|
Peroxisome |
11 |
IDH1、ACOX1、CAT、ACAA1、PEX3、PEX7、PMP34… |
|
|
Phagosome |
11 |
ATPeV1D、ATPeV1G、RAC1、RAB… |
|
|
Signal transduction |
Plant hormone signal transduction |
13 |
PTI1、AUX1、TIR1、GH3、GID1、SNRK2、BSK、BIN2、CTR1… |
|
Phosphatidylinositol signaling system |
6 |
PI4KB、PIP5K、DGK、PTEN、CALM |
|
|
Metabolism of terpenoids and polyketides |
Terpenoid backbone biosynthesis |
6 |
atoB、SDS、chlP、DHDDS、FLDH |
|
Membrane transport |
ABC transporters |
5 |
ABCB |
|
Lipid metabolism |
Glycerophospho lipid metabolism |
18 |
DGK、HM13、plcC、PRAF1、LYPLA2… |
|
Ether lipid metabolism |
13 |
PLDa1、PRAF1、LPCAT、LPT1… |
|
|
alpha-Linolenic acid metabolism |
9 |
EPT1、EIF3D、ACAA1、OPCL1、HPL… |
|
|
Glycerolipid metabolism |
8 |
ALDH、DGK、galA、AGPAT9、AGPAT8、LPT1… |
|
|
Fatty acid degradation |
7 |
ALDH、atoB、ACOX1、ACAA1... |
|
|
Steroid biosynthesis |
6 |
DHCR7、CAS、CYP51、SMT2、CPI1、CYP710A |
|
|
Environmental adaptation |
Plant-pathogen interaction |
18 |
CALM、HSP90B、CDPK、PTI1、LRR-RLK、PTI1、CML、RPM1… |
FP = the R. glutinosa
first planted in the field; SP= the R. glutinosa replanted in the same field continuously
%20337-348_files/image006.jpg)
Fig. 3: GO enrichment analysis of differently expressed membrane
proteins (a) up-regulated expressed membrane
proteins in replanted R. glutinosa, (b)
down-regulated expressed membrane proteins in replanted R. glutinosa
In this study, we discovered 10 up-regulated LRR-RLKs proteins closely related to the
immune system in the SP R. glutinosa.
Through the evaluation on the receptor kinase activity of these LRR-RLKs, we were able to assess the
invasion degree of pathogenic microorganism in the replanting soil. LRR-RLKs were important membrane
proteases that activated the phosphorylation of intracellular kinase domains through the specific binding of extracellular
signal-receiving sites to extracellular molecules, thereby accomplished transmembrane signal transduction. LRR-RLKs play an essential role in
pathogen infection in plants. In order to further confirm the role of LRR-RLKs
in the formation of replanting
stress, we infected the roots of R. glutinosa
plants with F. oxysporum, an SP R. glutinosa rhizosphere-specific
pathogenic fungi isolated by our research team in previous studies. As a
result, we found that F. oxysporum significantly
induced the up-regulated expression of LRR-RLKs
genes (Unigene7473_All, CL4884.Contig2_All and CL7625.Contig2_All). The expression of these genes was
consistent with our previous studies (Fig. 5).
Discussion
Plant cell membranes
play the important role in sensing %20337-348_files/image008.png)
external signals, dividing
organelles, ion exchange, and responding to %20337-348_files/image010.jpg)
Fig. 5:
LRR-RLKs expression analysis in FP and SP R.
glutinosa (a) Root electron microscopy
analysis of R. glutinosa
under F. oxysporum
(FO) infection, (b) Key LRR-RLKs expression analysis in FP and SP
R. glutinosa,
(c) Key LRR-RLKs expression analysis in FP and the R. glutinosa infected by F. oxysporum
(FO)
LRR-RLKs =
Leucine-rich repeat receptor-like kinases; FO= R. glutinosa infected by F. oxysporum
FP = the R. glutinosa
first planted in the field; SP= the R. glutinosa replanted in the same field continuously
external stresses. Therefore, the membrane system was important to survive of plants and the important channel for communicating
with environment (Liu et al. 2014; Sun and Wang 2009). Replanting disease mainly
caused by the change of soil physical and chemical properties, allelochemical
autotoxicity, and the imbalance of rhizosphere microbials (Lin et al. 2011, 2015; Wu et al. 2015; Zhang et al. 2018). The root membrane system may be plays
the important role in sensing and responding to replanting stress.
The study on the key
membrane proteins in response to replanting stress is thus important for analyzing the formation
mechanism of replanting disease in R. glutinosa.
After removing the non-membrane proteins, function analysis revealed
that the differential membrane proteins which related to immune system, calcium
signal response, and reactive oxygen species (ROS) metabolism were up-regulated
in the SP R. glutinosa.
In this study, 10 LRR-RLKs proteins
closely related to the immune system were up-regulated in the SP R. glutinosa. LRR-RLKs are the important membrane proteins in response to
pathogen infection, and usually play the important role in plant defense
response, growth and development, and hormone regulation (Osakabe et al. 2013; Wu and Zhou 2013; He and Wu
2016). LRR-RLKs receptor proteins were induced under various stress
conditions (such as pathogen infection, high salt, abscisic acid or injury, etc.) (Jung et al.
2004; Laura et al. 2009; Yang et al. 2012). Previous studies found that the rhizosphere microbes in SP R. glutinosa largely changed from
bacterial to fungal, which led to proliferation of large number of pathogenic
microorganisms (Chen et al. 2008; Lin et al. 2011). Numerous
studies have confirmed that the microecological imbalance in rhizosphere
mediated by allelochemical
autotoxins promoted the formation of replanting disease (Qiu et al. 2010; Li et al. 2012a, b; Zhao et al. 2016). Fusarium oxysporum, a
rhizosphere-specific pathogenic fungi of SP R. glutinosa isolated by our research team in previous studies (Li et al. 2016). To
further confirm the role of LRR-RLKs
in the SP R. glutinosa, R. glutinosa plants were inoculated in vitro using F. oxysporum. F. oxysporum significantly induced the up-regulation of LRR-RLKs genes (Unigene7473_All, CL4884.Contig2_All and CL7625.Contig2_All), which further confirmed the
relationship between the rhizosphere pathogen in SP R. glutinosa and the death of replanting plants.
Replanting stress induced membrane peroxidation in root tissue cells of
R. glutinosa. The immune response
triggered by pathogenic microbes rapidly induced downstream signaling
transduction including calcium signaling, ROS and other signaling pathways,
which further activate the expression of resistance genes (Poltronieri 2017;
Zhou and Zhang 2020). In plants, active oxygen was involved in the earlier
response to both biotic and abiotic stresses. Stresses could significantly
induce the production of various forms of ROS, such as superoxide, hydrogen
peroxide (H2O2), and hydroxyl radicals (Omar et al. 2020). The massive accumulation of ROS destroyed the
redox balance in plant cells and resulted in the oxidative damage to cellular
biomolecules, such as lipids, proteins, and nucleic acids (Cheng et al. 2013). Plants eliminated
excessive ROS mainly through antioxidant enzymes (such as superoxide dismutase,
peroxidase, and catalase), reduced lipid peroxidation, improved cell
metabolism, and ultimately alleviated the oxidative damage caused by stress. In
this study, seven types of peroxisome-related proteins were identified, of
which, there were three POD and two ACOX1 were
significantly down-regulated in replanted R. glutinosa. In plants, ACOX1
catalyzes acyl-CoA combined with oxygen, and formed trans-2, 3-dehydroacyl-CoA
and H2O2. In addition, in cellular membrane,
the phospholipase D were found to induce H2O2 biosynthesis.
For example, Qu et al. (2014)
discovered that the up-regulated expression of the PLD gene in Vicia faba root tips and Arabidopsis thaliana could induce
stomatal closure and H2O2 production (Qu et al.
2014). More importantly, Phospholipase D (PLD, EC 3.1.4.4) is
a type of lipolytic enzyme that hydrolyzes lecithin, phosphatidylethanolamine,
and phosphatidylglycerol under the induction of Ca2+, and αPLD
also hydrolyzed phosphatidylinositol diphosphate under acidic or Ca2+stimulation
(Rahier et al. 2016). Previous studies have found that calcium signaling
deeply involved in the formation of R.
glutinosa replanting disease (Yang et al. 2015). Furthermore, some studies on replanting disease in other plants
demonstrated that calcium signaling play the important role in response to
allelochemical stress (Chi et al. 2011). Therefore, calcium signaling-related proteins might
participate in the replanting disease as the downstream of receptor protein
kinases. The up-regulated expression of the PLD proteins and cellulose synthase
in the SP R. glutinosa root cells
might lead to the lipid peroxidation of the cell membrane and disruption of
cell membrane structure. Overview, the differential expression of the
identified enzymes related to H2O2 synthesis was
up-regulated in the SP R. glutinosa
root cells, while catabolism decreased, which led to oxidative stress in the
root cells. Excess H2O2 in the cell oxidized cell
membrane structure and destroyed biofilm structure.
In conclusion, based on the extraction of membrane
proteins and iTRAQ technology, two important molecular events that occurred in
cellular membrane of replanted R.
glutinosa including the imbalance of ROS metabolism and immune response,
were identified in this study. More importantly, this cellular process has
obviously proved to relate to the formation of replanting
disease in previous studies (Li et al. 2017; Chen and
Fluhr 2018). When replanted R. glutinosa faced the complex environment factors in rhizosphere, the proteins located in cellular membrane
were often first activated and respond to stress stimulus. At the same time, some
critical cellular process including the ubiquitin-mediated proteolytic enzymes,
RNA transporters, phytopathogenic interacting proteins, and dicarboxylic acid
metabolism-related proteins were significantly regulated in replanted R. glutinosa. Formation of replanting disease
was induced by multiple stress factors that mainly consisted of allelochemicals,
microbes in rhizosphere and their interactions (Zhao et al. 2015; Wu et al. 2017). Replanting stress was a complex stress, which caused severe
osmotic damage and membrane peroxidation in the root cells of the replanted R. glutinosa. Therefore, the response
process mediated by replanting stress may also be a cellular process induced by
the interaction of multiple proteins. This study confirmed the key damage
events induced by replanting stress through the analysis of differential
membrane proteins between FP and SP R. glutinosa,
although
the specific regulatory relationships of these differential membrane proteins
and their signal transduction processes will still require further experimental
verification, our findings provide the data foundation for
exploring the formation mechanism of replanting disease.
Conclusion
This study screened a suitable method for extracting membrane proteins
from the root tissue of R. glutinosa.
This extract method could highly effectively extract the membrane proteins from
R. glutinosa root tissue, the content
of membrane proteins reached 61.15%. The extracted membrane proteins were
located in the membrane structures of various organelles, and had the rich varieties.
Based on this, the differentially expressed membrane proteins in the root
tissues of FP and SP were identified. When SP R. glutinosa plants faced the complex environment factors in
rhizosphere, the stability of membrane structure was damaged, root cells formed
the cell signal transduction through the endocytosis by phagosomes,
transmembrane transport metabolism (dicarboxylic acid metabolism), proteins
export, and related metabolic regulation processes. Root tissue cells of SP induced
the external environmental signals, activated corresponding metabolic processes
and changed the process of protein transcription, translation and folding by
regulating the expression level of membrane proteins, and eventually led to the
replanting disease of R. glutinosa. These
results provided the theoretical basis for completing signal transduction
pathway of replanted R. glutinosa and
elucidating the key mechanisms associated with replanting stress.
Acknowledgements
This study was supported by the National Natural
Science Foundation of China (Grant No. 81573538, 81503193, 81603243
and 81403042)
and the Key Scientific Research Project of the higher Education
Institutions of Fujian Province of China (No. JK2015013).
Author Contributions
FJF, CYY and SYZ designed and performed the
research, analyzed data and wrote the manuscript. FJF, MJL,
SQC, LG, BZ and
AGC participated to analyze
data and helped in drafting the manuscript. ZYZ contributed to the
interpretation of results and coordinated the study. All authors have read and
approved the final draft.
Conflicts of Interest
Authors declare no conflict of interest.
Data Availability
Data presented in this study
will be available on a fair request to the corresponding author.
Ethics Approval
Not
applicable in this paper
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