Abstract
Plant growth promoting microbe assisted phytoremediation is considered
a more effective approach to rehabilitation than the single use of
plants, but underlying mechanism is still unclear. In this study, we
combined transcriptomic and physiological methods to explore the
mechanism of plant growth promoting microbe Trichoderma citrinoviride
HT-1 assisted phytoremediation of Cd contaminated water by Phragmites
australis. The results show that the strain HT-1 significantly promoted
P. australis growth, increased the photosynthetic rate, enhanced
antioxidant enzyme activities. The chlorophyll content and the activity
of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and
ascorbate peroxidase (APX) were increased by 83.78%, 23.17%, 47.60%,
97.14% and 12.23% on average, and decreased the content of
malondialdehyde (MDA) by 31.10%. At the same time, strain HT-1 improved
the absorption and transport of Cd in P. australis, and the removal
rate of Cd was increased by 7.56% on average. Transcriptome analysis
showed that strain HT-1 induced significant up-regulated the expression
of genes related to oxidative phosphorylation and ribosome pathways,
and these upregulated genes promoted P. australis remediation
efficiency and resistance to Cd stress. Our results provide a
mechanistic understanding of plant growth promoting microbe assisted
phytoremediation under Cd stress.
Keywords: Phytoremediation, Plant growth promoting microbe,
Plant–microbe association, Cd contaminant, Mechanism
Introduction
In recent decades, the improper disposal of industrial and domestic
waste, the excessive use of agricultural chemicals and the unreasonable
discharge of sewage from human activities, such as metal mining and
smelting have caused various heavy metal pollutants to enter the water.
This has resulted in the water pollution [[39]1]. Among all heavy
metals, cadmium (Cd) has been intensively studied due to its high
mobility and solubility (bioavailability), as well as its strong
cumulative toxicity [[40]2, [41]3]. As a typical toxic heavy metal, Cd
is one of the most harmful and ubiquitous water environmental
pollutants [[42]4], which has significant toxic effects on plants
[[43]5, [44]6]. At the same time, Cd is classified as a class I
carcinogen, which poses huge health risks to the human body as it
passes through the food chain [[45]7, [46]8]. At present, the use of Cd
in industry continues to increase, making it an escalating hazard
factor [[47]9]. Therefore, remediation of water contaminated with heavy
metals, particularly Cd, is of great importance for ensuring
environmental safety and human health, and is also very necessary and
urgen.
Traditional Cd pollution control technologies include precipitation
method, ion exchange method, membrane separation method, adsorption
method, etc. [[48]10]. However, these physical and chemical remediation
methods often encounter issues such as low efficiency in remediation,
high economic costs, and the potential for secondary pollution
[[49]11]. In the past decade, plant remediation has gradually become a
trend due to its economic, aesthetic and environmental protection
characteristics [[50]12]. A previous study reported that Pistia
stratiotes has shown promising remediation effects on water
contaminated with Cd [[51]13]. Celosia argentea Linn, Populus deltoids
and Salix viminalis were found to be effective in remediating
Cd-contaminated soil [[52]14–[53]16]. However, natural factors such as
environment, climate and the stress of heavy metals on plants have
limited the scope and effect of phytoremediation. Therefore, it is
necessary to enhance the exogenous reinforcement methods in order to
achieve the desired heavy metal remediation effect in the real
environment. Studies have found that the interaction between plants and
beneficial microorganisms, especially plant growth-promoting microbes
(PGPM) can significantly improve the remediation efficiency of plants
to heavy metal Cd [[54]17–[55]19]. Kamran et al. isolated a plant
growth-promoting bacteria (PGPB) Pseudomonas putida from the heavy
metal contaminated soil and found that bacterial inoculation increased
biomass of Cd hyperaccumulator plant Eruca sativa by up to 33%, and Cd
uptake increased by 29% [[56]20]. Ma et al. isolated a PGPB
Achromobacter piechaudii from the stem of hyperaccumulator Sedum
plumbizincicola, which significantly increased the bioavailability of
Cd, Zn and Pb, promote plant growth and enhanced plant uptake of Cd, Zn
and Pb [[57]21]. Wu et al. inoculated PGPB Pseudomonas fluorescens
isolated from the stems of Sedum alfredii into plants, which
significantly increased root biomass and Cd accumulation in plants,
with differences of 1.82 times and 3.04 times compared with the
control, respectively [[58]22]. However, so far, the key processes and
mechanisms of PGPM synergistic remediation of heavy metal pollution
have not been fully understood, especially at the molecular level.
Phragmites australis is a perennial graminaceous plant with high
biomass productivity [[59]23]. It is widely distributed throughout the
world and can survive in acidic conditions. In recent years, extensive
research and application have found that compared with other wetland
plants, P. australis is better at accumulation of some heavy metals
[[60]24]. Bragato et al. discovered that riparian wetland P. australis
opens the root-leaf transport system at the end of the growing season,
transferring heavy metals from the root system to the aging leaf
tissue, and removing heavy metal toxicity from the body through leaf
apoptosis [[61]25]. Bernardini et al. found that under high
concentrations of Zn and Pb in hydroponics, the physiological activity
of photosynthetic organs in P. australis was inhibited, but the roots
had a better ability to enrich heavy metals [[62]26]. In summary, P.
australis can be used to assess heavy metal pollution in wetlands and
it is an excellent plant for mitigating heavy metal pollution in water.
Trichoderma citrinoviride HT-1 was isolated from the root of Rheum
palmatum in the early stage of our research group. It has strong
vitality and outstanding colonization ability. T. citrinoviride HT-1
can produce IAA and siderophores to promote plants growth, and has good
inhibitory effect on many plant pathogens [[63]27]. In addition, the Cd
tolerance of strain HT-1 was determined, the results showed that strain
HT-1 has good Cd tolerance. In this study, physiological and
transcriptomic methods were used to explore the mechanism of
Trichoderma citrinoviride HT-1 improve the tolerance and repair
efficiency of P. australis to Cd. The purpose of this study is to (a)
explore the mechanism of strain HT-1 enhances the Cd tolerance of P.
australis; (b) the accumulation and transport of Cd in P. australis
plants under the treatment of strain HT-1 were analyzed; (c) identify
differentially expressed genes (DEGs) and their key pathways; and (d)
reveal the molecular mechanism of plant growth-promoting microbes to
improve the tolerance and repair ability of P. australis to heavy
metals. This study will provide a strong scientific basis for the
research and application of PGPM-P. australis combined remediation of
heavy metal pollution in water.
Materials and methods
Plant pre-culture
The seeds of P. australis were collected from Lanzhou Botanical Garden,
China (103^◦42′16.99″ E, 36^◦07′8.11″ N, 1583 m). Seeds were
surface-sterilized with 2% (v/v) NaClO solution for 20 min, then washed
with deionized water, and germinated on a petri dish (darkness at 25
℃). After seed germination, the seedlings were sowed in 5 L plastic
pots (43 × 19 cm, 12 seedings per pot) filled with water containing 1/4
Hoagland nutrient solution, which was sterilized at 121 ℃ for 2 h. The
temperature throughout the growth process was maintained at 25 ± 1 ℃ on
a 12 h light/12 h dark cycle, and the water was replenished every two
days.
Fungal strain culture
In this study, T. citrinoviride HT-1 (Accession number: [64]MT781604.1)
was preserved in the Microbial Collection of College of Life Science,
Northwest Normal University. The fungal isolates were maintained on PDA
medium (potato dextrose agar) at 4 ℃. The strain were resuscitated at
room temperature for 1 h and then inoculated on PDA plates for 7 d
under 28 °C 16 h light/8 h dark cycle. Conidial suspensions were
harvested from the PDA mediums through using 2 mL sterile water, and
then diluted to 10^7 spore/mL for subsequent experiments [[65]27].
Experimental design
After 8 weeks of seed germination, P. australis plants with uniform
growth were selected and placed in a 5 L plastic pot (36 plants per
pot). A total of 30 pots were prepared for ten different treatments,
each treatment contained three replicates: 0 mg/L Cd (sterile water);
0 mg/L Cd (sterile water) + strain HT-1; 5 mg/L Cd; 5 mg/L Cd + strain
HT-1; 10 mg/L Cd; 10 mg/L Cd + strain HT-1; 15 mg/L Cd; 15 mg/L
Cd + strain HT-1; 20 mg/L Cd; 20 mg/L Cd + strain HT-1; CdCl[2] was
used as the Cd source, which was added only once at the beginning of
the treatment. After 24 h of Cd treatment, the strain HT-1 conidial
suspension (10^7 spore/mL) was inoculated into plants of the
inoculation group at 5 mL/plant, the non-inoculation group was added
with the same amount of sterile water. After 4 weeks, the plants were
harvested for subsequent experiments.
Cd uptake and translocation effect of P. australis
The treated P. australis were cleaned and divided into roots, stems and
leaves for drying. 0.1 g of each plant sample was placed in a microwave
digestion tube and treated with 5 mL nitric acid (HNO[3]) and 1 mL of
30% hydrogen peroxide (H[2]O[2]) until completely digested using
microwave digestion instrument. The digestion liquid was filtered and
transferred to a 25 mL volumetric flask and diluted to volume with 1%
nitric acid. 50 mL hydroponic solution of each treatment group was
filtered with 0.22 μm filter membrane. Cd concentrations were then
determined with a flame atomic absorption spectrophotometer (AA7800,
Shimadzu, Japan). Three biological replicates were randomly selected
from each treatment.
The bioconcentration factor (BCF), translocation factor (TF) and Cd
removal rate were calculated to determine the Cd bio-accumulation and
the potential capacity of phytoremediation [[66]28, [67]29].
[MATH: BCF=Cd
concentration inplantCd concentration
inwater :MATH]
[MATH: TF=Cd
concentration inshootCd concentration
inroot :MATH]
[MATH: Cdremovalrate%=Cdinitialconcentrationinwater-CdconcentrationafterplantadsorptionCdinitialconcentrationinwater :MATH]
Growth index and physiological index of plants
The shoot length, root length, fresh weight of each sample were
immediately measured after 28 days of exposure to Cd stress
environment. Dried at 60 ^◦C for a week, and weighed [[68]30].
The roots of P. australis were cleaned 3–5 times by deionized water.
Root morphological traits were scanned by a root scanner (V700 PHOTO,
Epson, Japan), and WinRHIZO™2003b software (Regent Instruments, QC,
Canada) was used to analyze total root surface area (SA) [[69]31].
The root activity of P. australis was tested according to the triphenyl
tetrazolium chloride (TTC) method [[70]32]. A total of 0.1 g fresh P.
australis roots were cut into pieces and immersed in 0.6% (w/v) TTC
solution (TTC was dissolved in phosphate buffer at pH7.0) for 24 h at
30 ^◦C in the dark. Then, the roots were rinsed twice, the water from
the roots’ surfaces was removed, and the roots were immersed in 95%
(v/v) ethanol for 30 min at room temperature. The absorbance values of
the extraction solutions were tested by a spectrophotometer (Hitachi
U3010, Tokyo, Japan) at 485 nm.
0.1 g leaves of the same part of each treated plant were collected.
With 95% ethanol as solvent, grinded into homogenate on ice, filtered
and diluted to 25 mL. The absorbance at 665, 649 and 470 nm were
recorded (Hitachi U3010, Tokyo, Japan), with 95% ethanol as the blank,
the whole experiment was carried out under dark conditions. The mean
value was derived from three repeats. The chlorophyll content was
tested by using the procedure of Li et al. [[71]33].
The first fully expanded leaf at the top of the plant was used for
measuring leaf gas exchange traits, including stomatal conductance
(Gs), net photosynthesis rate (Pn), intercellular CO[2] (Ci) and
transpiration rate (Tr). A portable infrared gas exchange analyzer
(GFS-3000, WALZ, Germany) was used, and measurements were taken between
9:00 h and 12:00 h Beijing time. The data were taken under
1200 μmol m^−2 s^−2 light intensity, 25 ^◦C leaf temperature, and
440 μmol mol^−1 CO[2] concentration [[72]33]. Leaves from six randomly
selected seedlings from each treatment were clamped into the leaf
chamber and measured after the net photosynthetic rate readings
stabilized.
Malondialdehyde (MDA) was measured by the method of Mbonankira et al.
[[73]34]. Cold 10% trichloroacetic acid was added into a 0.1 g leaf
sample and centrifuged at 4000 r/min for 10 min. Then, 2 mL of 0.6%
thiobarbituric acid were added to the sample and incubated in a 100 ^◦C
water bath for 15 min. The supernatant was quickly cooled and
centrifuged again. OD values were determined using an ELISA reader at
600 nm and 532 nm, respectively.
0.1 g leaf sample was ground in 1 mL extraction buffer (50 mM PBS,
pH = 7.8), 1 mM EDTA-Na[2], and 0.1% PVP) and centrifuged at 4 ^◦C for
20 min at 12,000 rpm. The supernatant was used to determine the
activities of superoxide dismutase (SOD), peroxidase (POD), catalase
(CAT) and ascorbate peroxidase (APX). The activity of SOD was estimated
using the method of Giannopolitis and Ries [[74]35]. The activities of
POD and CAT were assayed in accordance with Maehly and Chance’s
[[75]36]. The activity of APX was measured following the method of
Nakano and Asada [[76]37]. Each treatment was repeated at least three
times [[77]38].
Transcriptome analysis
The P. australis root of 15 mg/L Cd + strain HT-1 treatment and only
15 mg/L Cd treatment were frozen in liquid nitrogen, and three
biological replicates for transcriptomic analysis. Total RNA was
extracted from samples using TRIzol reagent. (Invitrogen, Thermo Fisher
Scientific Inc., Waltham, MA, USA). The quality of RNA was determined
by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto,
CA, USA) and a NanoDrop (Thermo Fisher Scientific Inc., Waltham, MA,
USA). The RNA integrity numbers (RINs) of RNAs > 7 were selected to
build the library and the cDNA library was sequenced on the Illumina
sequencing platform by Personalbio Technology Co., Ltd. (Shanghai,
China). The low-quality data and adaptor sequences in the original data
were filtered out to ensure the accuracy of the data. De novo assembly
was performed togenerate transcripts according to the Trinity method
[[78]39]. HTSeq (v0.6.1) was used to estimate the gene and isoform
expression levels from paired-end clean data [[79]40]. Differentially
expressed genes (DEGs) were analyzed using DESeq2 (V1.6.3) in the
Bioconductor software package [[80]41]. Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway enrichment analysis of DEGs was performed using
KOBAS [[81]42]. Gene Ontology (GO), an international standardized gene
function classification, was performed using the BLAST2 GO tool
[[82]43].
qRT–PCR analysis
An amount of 3 µg of purified total RNA was used as a template for
first-strand cDNA synthesis using PrimeScript^TMRT reagent Kit with
gDNA Eraser (TaKaRa, Kyoto, Japan) for qPCR. Several genes identified
by RNA-seq were selected for amplification using SYBR Green qPCR.
Primers were designed using Sangon Biotech. Actin was used as a
reliable internal reference gene for judging the efficiency of the
RT-PCR system and the quality of RNA extracts (Table [83]1). qRT–PCR
was conducted in a 10 μl volume containing. Six biological replicates
were selected for measurement, the 2^−∆∆CT method was used to calculate
relative expression levels [[84]44, [85]45].
Table 1.
qRT-PCR primers used in this study
Gene Name Forward primer(5'-3') Reverse primer(5'-3')
IAA GGAGGTCTGTACGTGAAGGTGAG GAGGAGAAGCAGCCGAAGAGC
ERF
ATGGATGCGACCTTCCGAGATTC
ATGGATGCGACCTTCCGAGATTC
TCACCGAGTCACCCACAAACAC
APX CGATGATGATGTCCACGCAGTTG CCGATCCGATCCAGCATTCCAG
PCS TTTACACTTCTTCTTGGCTGCTTGG ACGCGACCTTGGCTATCAACTAC
MYB CGAGGAGGATGCGAGACTGTTAG GCCAAGTTGTTCACATAGCGATCTC
ATP CTGCTTCTGGGTGCCCTTGG ATCGTCGCCGTTGTCCGTATC
HMA GCGGCAAGAGCGTTAATCACTG
AAGAGTTTCTGACCTTCCAGTAGCC
AAGAGTTTCTGACCTTCCAGTAGCC
ABC TACTGGTGCTGCTAAGATGGATGC GGAGGATGTGCTCTCTGGTTTGG
ZIP CACGCCGCAGCAAATCCAAG CTCTCCAGGTTGAGCCGCTTG
WRKY
CTGGAGCAGCAGCAGCAGAC
CTGGAGCAGCAGCAGCAGAC
GCGGCAGGAGCAAGGATGAC
CACGCCGCAGCAAATCCAAG
Actin CGAGCACGGTATTGTTAGCAACTG CGCCTCAGTCAGCAGCACAG
[86]Open in a new tab
Statistical analysis
All data were analyzed by SPSS26.0 software for variance (one-way,
ANOVA) and Duncan's multiple range test (P < 0.05). Columns were
constructed by using Origin 2023 (Origin Software, Northampton, MA,
USA).
Results
Effects of the growth of P. australis seedlings by the inoculation of strain
HT-1 under Cd stress
In the present study, the biomass of P. australis decreased with
increasing CdCl[2] concentration. However, strain HT-1 treatments
significantly increased the shoot length, root length, fresh weight,
dry weight and the total root surface area of plants (Figs. [87]1 and
[88]2). Among them, after inoculation of strain HT-1, the increase of
P. australis root length and the total root surface was the most
significant under the concentration of CdCl[2] was 15 mg/L, the growth
rates were 22.79% and 65.44% respectively compared with the
non-inoculated plants (Fig. [89]1A, E). The increase of the shoot
length, fresh weight and dry weight of P. australis were the most
significant under the the concentration of CdCl[2] was 20 mg/L, which
were increased by 28.62%, 78.05% and 100.66% respectively compared with
the non-inoculated plants (Fig. [90]1B, C, D).
Fig. 1.
[91]Fig. 1
[92]Open in a new tab
Effects of strain HT-1 on the growth of P. australis in different
concentration of Cd, A shoot length, B root length, C fresh weight, D
dry weight, E total root surface area. Values are mean ± SD (n = 15
plants). Different letters abovethe bars indicate the differences are
significant at P < 0.05
Fig. 2.
[93]Fig. 2
[94]Open in a new tab
Effects of strain HT-1 on the growth of P. australis in different
concentration of Cd, A 0 mg/L Cd (sterile water), B 0 mg/L Cd (sterile
water) + strain HT-1, C 5 mg/L Cd, D 5 mg/L Cd + strain HT-1, E 10 mg/L
Cd, F 10 mg/L Cd + strain HT-1, G 15 mg/L Cd, H 15 mg/L Cd + strain
HT-1, I 20 mg/L Cd, J 20 mg/L Cd + strain HT-1
Effects of Cd accumulation and transport in P. australis by the inoculation
of strain HT-1
We measured Cd of the enrichment factor (BCF), transport factor (TF)
and plant removal rate. As shown in Table [95]2, When the concentration
of CdCl[2] was 20 mg/L, the content of Cd^2+ was the highest in leaf,
stem, root, which were 15.29 mg/kg, 16.36 mg/kg and 73.24 mg/kg,
respectively. The strain HT-1 significantly promoted the absorption
Cd^2+ of P. australis. Under different Cd treatment concentrations, the
Cd^2+ content in roots, stems and leaves of inoculated was
significantly higher than that of non-inoculated plants. When the
concentration of CdCl[2] was 5 mg/L, the Cd^2+ content in the leaves
and stems of the inoculated increased most significantly, and the
growth rates were 408.10% and 235.54% compared with the non-inoculated
treatment, respectively. When the concentration of CdCl[2] was 15 mg/L,
the Cd^2+ content of roots increased most significantly with the
inoculation of strain HT-1, the growth rate was 20.13% compared with
the non-inoculated treatment.
Table 2.
The effect of strain HT-1 inoculation on Cd uptake and transport in P.
australis
Cd treatment (mg/L) Leaf Cd
content
(mg/kg) Stem Cd
content
(mg/kg) Root Cd
content
(mg/kg) BCF TF Cd removal rate (%)
0 non-inoculated ND ND ND ND ND ND
inoculated ND ND ND ND ND ND
5 non-inoculated 3.62 ± 0.01 h 7.57 ± 0.11 h 65.23 ± 0.54 h
15.28 ± 0.09 b 0.17 ± 0.0029 h 74.44 ± 0.13 e
inoculated 18.39 ± 0.04 d 25.40 ± 0.21 d 71.85 ± 0.57 e 23.13 ± 0.15 a
0.61 ± 0.0028 d 87.32 ± 0.97 d
10 non-inoculated 8.90 ± 0.02 g 12.13 ± 0.32 g 68.40 ± 0.12 g
8.94 ± 0.05 e 0.31 ± 0.0077 g 87.13 ± 0.31 d
inoculated 19.10 ± 0.06 c 38.40 ± 0.33 a 76.95 ± 0.28 c 13.44 ± 0.03 c
0.75 ± 0.0117 c 88.44 ± 0.01 d
15 non-inoculated 9.88 ± 0.02 f 14.79 ± 0.02 f 70.01 ± 0.18 f
6.31 ± 0.01 g 0.35 ± 0.0008 f 90.83 ± 0.25 c
inoculated 38.74 ± 0.03 a 37.22 ± 0.03 b 84.10 ± 0.07 a 10.67 ± 0.01 d
0.90 ± 0.0019 a 96.56 ± 0.04 a
20 non-inoculated 15.29 ± 0.17 e 16.36 ± 0.18 e 73.24 ± 0.13 d
5.24 ± 0.02 h 0.43 ± 0.0060 e 92.87 ± 0.14 b
inoculated 36.90 ± 0.06 b 31.52 ± 0.03 c 82.83 ± 0.15 b 7.56 ± 0.00 f
0.83 ± 0.0043 b 97.63 ± 0.04 a
[96]Open in a new tab
Different lowercase letters in the same column indicated significant
difference between treatments (P < 0.05)
As showed in Table [97]2, it can be seen that the BCF of P. australis
decreases with the increase of CdCl[2] concentration in water,
indicating that P. australis have better enrichment ability in low Cd
environment. After inoculation with strain HT-1, the BCF of Cd in P.
australis increased significantly. The maximum BCF was observed in P.
australis at 15 mg/L CdCl[2], which was increased by 69.10% compared
with the non-inoculated treatment. At the same time, the TF of P.
australis increased with the increase of CdCl[2] concentration in
water, indicating that with the transport capacity of plants to heavy
metals gradually increased with the increase of its concentration,
which led to more heavy metals transported from roots to shoots, and
was beneficial to plants to absorb more heavy metals. After inoculated
strain HT-1, the transport coefficient of plants in each treatment
group was significantly higher than that of non-inoculated, among them,
TF increased most significantly when the concentration of CdCl[2] was
5 mg/L, and the growth rate was 258.82% compared with non-inoculated
plants. In addition, with the increase of CdCl[2] concentration, the
removal rate of Cd in water by P. australis showed an increasing trend,
and the removal rate of Cd by plants was significantly increased after
inoculation with strain HT-1. When CdCl[2] was 5 mg/L, the inoculation
of strain HT-1 had the most significant effect on the Cd removal rate
of P. australis. Compared with the non-inoculated treatment, the growth
rate was 17.3%.
Physiological response of strain HT-1 to P. australis seedlings under Cd
stress
As shown in Fig. [98]3, with the increase of CdCl[2] concentration, the
root activity decreased significantly. Strain HT-1 treatments
significantly increased the root activity of P. australis. At the
concentration of CdCl[2] of 15 mg/L, the root activity of inoculated
with strain HT-1 had the highest growth rate of 44.57%. The difference
was significant at the 0.05 level (P < 0.05).
Fig. 3.
Fig. 3
[99]Open in a new tab
Effects of strain HT-1 on root activity of P. australis in different
concentration of Cd. Values are mean ± SD (n = 15 plants). Different
letters abovethe bars indicate the differences are significant at
P < 0.05
In this study, we measured the changes of photosynthetic pigment
contents in P. australis (Fig. [100]4). With the increase of CdCl[2]
concentration, the chlorophyll content (a + b) of plants decreased
significantly. Strain HT-1 treatments significantly increased the
chlorophyll content of P. australis. Among them, after inoculation of
strain HT-1, the increase of chlorophyll content was the most
significant under the concentration of 20 mg/L CdCl[2], the growth rate
was 110.25% compared with the non-inoculated plants (Fig. [101]4A). In
addition, with the increase of CdCl[2] concentration, the leaf gas
exchange attributes in P. australis, including net photosynthetic rate
(Pn), leaf stomatal conductance (Gs), intercellular CO[2] concentration
(Ci) and transpiration rate (Tr) decreased significantly. However,
strain HT-1 inoculation significantly increased Pn, Gs, Ci and Tr of P.
australis under CdCl[2] treatment (P < 0.05) (Fig. [102]4B, C, D, E).
These results indicate that strain HT-1 protects the P. australis
photosynthesis under heavy metal Cd stress.
Fig. 4.
[103]Fig. 4
[104]Open in a new tab
Effects of strain HT-1 on the photosynthesis of P. australis in
different concentration of Cd, A chlorophyll content, B net
photosynthetic rate (Pn), C leaf stomatal conductance (Gs), D
intercellular CO[2] concentration (Ci), E transpiration rate (Tr).
Values are mean ± SD (n = 15 plants). Different letters abovethe bars
indicate the differences are significant at p < 0.05
It can be seen that activities of antioxidant enzyme (SOD, POD, CAT and
APX) in P. australis leaves were significantly lower than control
seedlings with the increase of CdCl[2] concentration (Fig. [105]5B, C,
D, E). After inoculation with strain HT-1, the activities of SOD, POD,
CAT and APX at most were 0.33, 0.75, 1.13 and 0.20 times higher than
those of uninoculated plants, respectively, which were significantly
increased the activities of antioxidant enzymes (P < 0.05). In
addition, we found that the content of MDA in P. australis leaves
increased significantly with the increase of CdCl[2] concentration.
However, strain HT-1 treatments significantly redused the MDA content
of P. australis. Among them, after inoculation of strain HT-1, the
reduction of MDA content was the most significant under the
concentration of CdCl[2] was 5 mg/L, the reduction rate was 41.10%
compared with the non-inoculated plants (Fig. [106]5A).
Fig. 5.
[107]Fig. 5
[108]Open in a new tab
Effects of strain HT-1 on the antioxidant system of P. australis in
different concentration of Cd, A MDA content, B SOD content, C POD
content, D CAT content, E APX content. Values are mean ± SD (n = 15
plants). Different letters abovethe bars indicate the differences are
significant at p < 0.05
In summary, heavy metal cadmium seriously damaged the structure and
function of P. australis leaves and root tissues. Strain HT-1 can
alleviate the damage of Cd to plant photosynthetic system, improve the
antioxidant capacity of plants, and thus improve the tolerance of
plants to heavy metals. Combined with physiological and biochemical
analys8is, it can be found that when the concentration of CdCl[2] is
15 mg/L, the inoculation strain HT-1 has the most significant
improvement in the indicators of P. australis seedlings. At this time,
the strain exerts its best biological function. Therefore, we next
performed transcriptome analysis on the roots of P. australis seedlings
treated with 15 mg/L CdCl[2] to explore the molecular mechanism of
strain HT-1 improving P. australis to alleviate Cd toxicity in polluted
environment.
Unigene annotation and identification of DEGs
A total of 118,927,346 and 131,503,608 raw reads were obtained from the
inoculation treatment and non-inoculation treatment groups,
respectively. 117,143,178 (inoculation treatment) and 129,347,506
(non-inoculation treatment) clean reads were retained after assembly
(Table [109]3). Sequencing data were submitted to the NCBI
([110]https://www.ncbi.nlm.nih.gov/), accession number was
PRJNA1032252. A total of 518,426 unique sequences were annotated based
on blastx alignment searches of six public databases including GO,
KEGG, NR, eggNOG, Swiss-prot and Pfam. A total of 8070 genes were
identified, of which 2057 genes were up-regulated (|logFC|> 1) and 6013
genes (|logFC|> 1) were down-regulated (Fig. [111]6).
Table 3.
Summary of trimming and read mapping results of the sequences obtained
from P. australis roots treated without or with strain HT-1 inoculation
Sample Raw_reads Clean_reads
15 mg/L Cd (1) 40,758,436 40,177,876
15 mg/L Cd (2) 41,123,416 40,477,238
15 mg/L Cd (3) 37,045,494 36,488,064
15 mg/L Cd + strain HT-1 (1) 44,118,746 43,369,004
15 mg/L Cd + strain HT-1 (2) 39,453,560 38,754,802
15 mg/L Cd + strain HT-1 (3) 47,931,302 47,223,700
[112]Open in a new tab
Fig. 6.
Fig. 6
[113]Open in a new tab
Volcano plots of differentially expressed genes, A 15 mg/L Cd
treatment, B 15 mg/L Cd + strain HT-1 treatment
GO function and KEGG pathway enrichment analysis of the DEGs
GO analysis elucidated the specific biological functions of DEGs in the
two comparisons,which were classified in three ontologies (biological
processes (BP), molecular functions (MF) and cellular components (CC).
The top 10 significantly enriched GO terms concerning were shown in
(Fig. [114]7). The upregulated DEGs were mainly involved in catalytic
activity, structural constituent of the ribosome (MF), cellular
nitrogen compound biosynthetic process, peptide metabolic process and
translation (BP), extracellular region and ribosome (CC). The
downregulated DEGs were significantly enriched in strutural molecule
activity (MF), ribonucleoprotein complex, non-membrane-bounded
organelle and ribosome (CC), and translation (BP). GO analysis
indicated that strain HT-1 could effectively enhance such as the
catalytic activity, ribosome metabolism, translation, etc., and defense
mechanism of plants. So as to improve the Cd tolerance and enable plant
survival better in stressed environments.
Fig. 7.
[115]Fig. 7
[116]Open in a new tab
Chart summarizing the results of Gene Ontology (GO) enrichment analysis
All DEGs were assigned to the KEGG database for KEGG pathway enrichment
analysis. Research found that DEGs were significantly enrich in
metabolic pathways and biosynthesis of secondary metabolites. KEGG
enrichment analysis as scatter plots with the 20 most significantly
enriched pathways. Of which, the ribosome, endocytosis, limonene and
pinene degradation, protein processing in endoplasmic reticulum and
photosynthesis were the top five enrich pathways in upregulated DEGs
(Fig. [117]8). In contrast, the ribosome, oxidative phosphorylation,
arachidonic acid metabolism, citrate cycle and nitrogen metabolism were
the top five pathways in downregulated DEGs. KEGG analysis indicated
that strain HT-1 can regulated amino acid metabolism, nitrogen
metabolism, carbohydrate metabolism and strengthen signal transduction
to reduce the toxicity of Cd to plants.
Fig. 8.
[118]Fig. 8
[119]Open in a new tab
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of
differentially expressed genes (DEGs)
Transcription factors (TF) analysis
Changes in gene expression caused by strain HT-1 ultimately regulate
the response mechanism of P. australis roots to Cd stress. This
research examined 12,577 hypothetical TFs from 57 different families.
The top 10 TFs were bHLH (1204), NAC (973), MYB-related (954), ERF
(709), C2H2 (677), WRKY (643), bZIP (571), FAR1 (486), MYB (475), C3H
(469) (Fig. [120]9).
Fig. 9.
[121]Fig. 9
[122]Open in a new tab
Changes in gene expression
Verification of partial DEGs using RT-qPCR
To validate the reliability of RNA-Seq data, we selected 10 DEGs
related to plant growth, photosynthesis, antioxidant activities and
metal transport, including IAA (DN1943_c1_g1), ERF (DN7252_c0_g1), APX
(DN1099_c0_g1), PCS (DN1373_c0_g2), MYB (DN29668_c0_g1), ATP
(DN88929_c1_g1), HMA (DN139448_c0_g1), ABC (DN38393_c0_g1), ZIP
(DN19222_c0_g1) and WRKY (DN1823_c1_g1) (Table [123]4), quantitative
analysis of gene expression was detected using real-time quantitative
PCR (qPCR) examined them by qRT-PCR. The primers used for qRT-PCR are
listed in Table [124]4. As expected, the expression profiles of these
DEGs were consistent with the RNA-seq results (Fig. [125]10),
indicating the dependability of the RNA-Seq data.
Table 4.
The annotation of selected functional genes
Gene name Gene ID NR Annotation
IAA DN1943_c1_g1 Auxin-responsive protein
ERF DN7252_c0_g1 hylene-responsive transcription factor
APX DN1099_c0_g1 ascorbate peroxidase
PCS DN1373_c0_g2 Photosystem II protein
MYB DN29668_c0_g1 Transcription factor MYB3R-5
ATP DN88929_c1_g1 Plasma membrane ATPase
HMA DN139448_c0_g1 Cadmium/zinc-transporting ATPase
ABC DN38393_c0_g1 ABC transporter family
ZIP DN19222_c0_g1 homeobox-leucine zipper protein
WRKY DN1823_c1_g1 WRKY transcription factor
[126]Open in a new tab
Fig. 10.
[127]Fig. 10
[128]Open in a new tab
qRT-PCR assay of genes in P. australis
Discussion
As a non-essential element, the accumulation of trace amounts of Cd in
plants can result in dwarfing of plants, yellowing of leaves, slowed
root growth, and hindered overall plant growth [[129]46, [130]47].
Previous studies has shown that PGPM provides sufficient nutrients for
plants by producing siderophores, solubilizing P and fixing nitrogen,
and can also synthesize plant growth-regulating hormones to promote
plant growth and enhance resistance in heavy metal-contaminated
environments [[131]48, [132]49]. In our study, with the increase of
CdCl[2] treatment concentration, the growth of plants was significantly
inhibited. Plant growth was significantly improved after treatment with
strain HT-1. As a plant growth-promoting bacteria, strain HT-1 has the
function of producing IAA and siderophores, as well as antibacterial
and disease resistance [[133]28, [134]50]. Therefore, after inoculated
strain HT-1, it can directly lead to an increase in P. australis
biomass and indirectly promote plant growth by increasing plant
resistance to heavy metals.
The efficiency of remediation depends on the bioactivity of residual
heavy metals in the environment and their availability in plants
[[135]51, [136]52]. Apart from plant growth promotion, PGPM were proven
can also assist plant heavy metal uptakes and accumulation via
increasing their solubility and bioavailability [[137]53]. Wang et al.
resulted that PGPM inoculants enhanced oilseed Cd concentration and Cd
phytoextraction efficiency, and the soil Cd removal rate of PGPM
inoculated plants was 2.44 times higher than that of non-inoculated
plants [[138]54]. Asilian et al. inoculated Piriformospora indica
increased plant root Cd concentration and the uptake of maize
[[139]55]. Chen et al. reported that inoculating Sphingomonas SaMR12
significantly increased Zn uptake by S. alfredii was close to 23 fold
[[140]56]. In this study, inoculating strain HT-1 significantly
improved the absorption and transportation of Cd by P. australis, and
enhanced the removal rate of Cd in water. Our results show that
inoculation with strain HT-1 can effectively improve the remediation
efficiency of P. australis to Cd in water.
Plant growth depends on the cycle between the above-ground and
below-ground parts, with the above-ground part synthesizes the products
of photosynthesis into a carbon source and the below-ground part
continuously absorbs and transports water and nutrients [[141]46].
Therefore, the degree of root development and the strength of
photosynthesis can be used as a basis for evaluating plant growth
status [[142]57]. In our study, plant total root surface area and root
activity, the chlorophyll content, net photosynthetic rate, stomatal
conductance, intercellular CO[2] concentration and transpiration rate
of P. australis leaf showed that the inoculated treatment was better
than non-inoculated treatments. It indicates that strain HT-1 improves
the coordination between photosynthesis and root growth of P.
australis, thus promoting the physiological metabolism of plants.
Heavy metal Cd is an inducer of oxidative stress, which typically leads
to lipid peroxidation of plant cell membranes and generates a
significant amount of reactive oxygen species (ROS), resulting in
oxidative damage to plants [[143]52, [144]58]. In addition, Cd toxicity
also inhibits the photoactivation of photosystem II (PSII), which leads
to the destruction of chloroplasts in leaves and indirectly promotes
the production of ROS [[145]59, [146]60]. Unregulated ROS in plant
cells can disrupt cell integrity and function, leading to detrimental
effects on plant growth [[147]61]. Therefore, under cadmium stress,
plants activate their own antioxidant defense system and increase the
activities of antioxidant enzymes such as SOD, POD, APX, CAT and GR to
scavenge toxic free radicals and protect themselves from oxidative
stress [[148]62, [149]63]. In this study, with the increase of CdCl[2]
treatment concentration, the activities of antioxidant enzymes in P.
australis decreased significantly. However, the inoculated strain HT-1
could significantly increase the activities of four antioxidant
enzymes, thereby helping plants to alleviate Cd stress, which was
consistent with the results of Pan et al. and Raja et al. [[150]64,
[151]65], indicating that PGPM could change enzyme activity and
increase the ability of plants to resist oxidative stress. The
generation of ROS and the subsequent oxidative stress resulted in high
MDA content. MDA is a byproduct of lipid peroxidation that usually used
to assess the extent of cell membrane damage [[152]66]. High levels of
MDA can lead to a decrease in cell water content and membrane integrity
which negatively affects plant metabolic functions, yield and growth
[[153]67]. In our study, Regardless of whether Cd stress occurred or
not, inoculation with strain HT-1 significantly reduced the content of
MDA in P.australis leaves. This reduction was significantly different
from the non-inoculated treatments. It indicated that strain HT-1 could
alleviate damage to the plant cell membrane system and improve the
tolerance of P.australis to Cd. The result is consistent with the
research of Wang et al. [[154]68].
Cd stress can induce the expression of stress-related genes and
proteins [[155]69]. The effect of strain HT-1 also alters the
expression of plant genes and proteins [[156]28]. In previous studies,
RNA-seq sequencing has been extensively utilized to investigate the
gene and protein responses of gramineous plants, including rice
[[157]70], wheat [[158]31], and maize [[159]71] under heavy metal
stress. However, RNA-Seq analysis of P. australis under Cd stress is
limited. In this study, 8070 differentially expressed genes were mainly
involved in ribosome, amino acid metabolism and other pathways.
Therefore, we studied the DEGs from plant hormone signal transduction,
oxidative phosphorylation and plant-microorganism interaction.
Indoleacetic acid (IAA) promoted plant fixation of mineral elements,
which played a positive role in plant growth [[160]72]. As a main
transcription factor protein, ERF potentially involved in the growth
process of various plants [[161]73]. APX, a DEG associated with
antioxidant enzymes, plays an important r ole in plant antioxidant
defense system [[162]31]. As a subunit of photosystem II (PSII), PCS
participates in plant photosynthesis-related physiological activities,
and its expression level is positively correlated with plant
photosynthesis capacity [[163]74]. In our study, after inoculation with
strain HT-1, the expression levels of the above genes in P. australis
were significantly increased. This is similar to the findings of Liu et
al. who observed an increase in the expression of IAA and CAT genes in
plants after treating Cd-stressed wheat with silicon [[164]31]. The
results show that strain HT-1 can mediate the transduction of signaling
pathways, such as plant hormone synthesis and antioxidant defense
system. It can also promote the expression of related genes, thereby
alleviating oxidative stress and enhancing plant tolerance to cadmium.
In addition, this study also screened a large number of DEGs related to
Cd uptake and transport, including zinc ion transmembrane transporter,
ABC transporter and metal ion transmembrane transporter, as well as key
Cd transporters such as MYB, ATP and HMA. ZIP family transporters are
mainly present in the inner membrane system of plants and play an
indispensable role as Zn/Cd transporters [[165]75]. ABC transporters
can be strictly involved in plant metal transport [[166]76–[167]79]. In
this study, the treatment with strain HT-1 significantly affected the
expression of these genes, indicating that strain HT-1 enhanced the
repair capacity of P. australis for Cd by regulating the transporters
of heavy metals. Khan et al. also reported that the expression of a
large number of genes in the HMA family was significantly affected
after melatonin treatment of Cd-stressed cotton seedlings [[168]80].
This suggests that HMA may play a role in melatonin-induced relief of
Cd stress. In summary, the results of this study indicate that strain
HT-1 activates transcription factors through signal transduction, which
in turn triggers the expression of a series of functional genes. This
activation reduces the growth inhibition of P. australis caused by Cd
and ultimately enhances the plants' tolerance to Cd. The ability to
absorb and transport Cd has been greatly improved.
Conclusion
In this study, the effects of plant endophytic growth-promoting
microorganisms on Cd tolerance and repair ability of P. australis were
analyzed by physiological, biochemical and transcriptomic methods. We
found that strain HT-1 induced significant up-regulated the expression
of genes related to oxidative phosphorylation and ribosome pathways,
thereby increasing the growth rate, photosynthetic rate, antioxidant
capacity and Cd uptake and transport rate of P.australis, enhancing P.
australis remediation efficiency and resistance to Cd stress. This
study provides new insights for PGPM to improve plant tolerance to
heavy metals and remediation efficiency.
Acknowledgements