Plant Physiology and Biochemistry 197 (2023) 107661

Contents lists available at ScienceDirect

Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Transcriptome studies on cadmium tolerance and biochar mitigating

cadmium stress in muskmelon
Yuxuan Cheng a, 1, Lingzhi Qiu a, 1, Pingkai Shen a, Yunqiang Wang b, c, Junli Li a, *, Zhaoyi Dai b, c,
Meifang Qi a, Ying Zhou a, Zhengkang Zou a
a
School of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of Technology, Wuhan, 430070, PR China
b
Institute of Economic Crops, Hubei Academy of Agricultural Science, Wuhan, 430064, PR China
c
Vegetable Germplasm Innovation and Genetic Improvement Key Laboratory of Hubei Province, Hubei Academy of Agricultural Science, Wuhan, 430064, PR China

A R T I C L E I N F O A B S T R A C T

Handling Editor: K Kees Venema Cadmium pollution in agricultural soil is a great threat to crop growth and human health. In this research, with
1%, 3% and 5% biochar applied to control soil cadmium pollution, melon was selected to be the experimental
Keywords: object for physiological detection and transcriptome analysis, through which we explored the mechanism of
Soil cadmium pollution cadmium tolerance and biochar mitigating cadmium stress in muskmelon. Three set concentrations of biochar
Bioaccumulation
have a mitigative effect on muskmelon cadmium stress, and 5% biochar and 3% biochar respectively have the
Transcriptome
best and the worst alleviative effect. The alleviation of biochar to cadmium stress on muskmelon is primarily in
Muskmelon
Soil remediation the manner of inhibiting cadmium transfer, while the resistance of muskmelon to cadmium stress is through
Biochar activating phenylpropanoid pathway and overexpressing stress related genes. Under cadmium treatment, 11
genes of the phenylpropane pathway and 19 stress-related genes including cytochrome P450 family protein
genes and WRKY transcription factor genes were up-regulated, while 1%, 3%, 5% biochar addition significantly
downregulated 3, 0, 7 phenylpropane pathway genes and 17, 5, 16 stress-related genes, respectively. Genes such
as cytochrome P450 protein family genes, WRKY transcription factor genes, and annexin genes may play a key
role in muskmelon’s resistance to cadmium stress. The results show the key pathways and genes of cadmium
stress resistance and the effect of different concentrations of biochar in alleviating cadmium stress, which provide
a reference for the research of cadmium stress resistance in crops and the application of biochar in cadmium
pollution in agricultural soil.

1. Introduction
As one of the most toxic heavy metal contaminants, cadmium has
always been a major pollutant in agricultural soil, in the context of
cadmium contamination intensifying around the world (Zou et al.,
2021). Cadmium is difficult to degrade, so it can easily accumulate in the
soil. Especially in some closed, rain-washed crop planting environments,
the cadmium content in the soil tends to exceed the limit of 0.3 mg/kg
specified by the agricultural industry standard (NY 5294–2004) (Sun
et al., 2020). In the root environment of some crops, only a few mole of
cadmium can cause damage to the root system of crops, especially the
reduction of the crops photosynthetic efficiency and oxidative stress
response of crops (Cengiz et al., 2020). Most crops such as rice, wheat,
sweet potatoes and other crops planted on pollution soil will be subject
to cadmium stress (Zhang et al., 2021; Lijuan et al., 2021; He et al.,
2021), following the crop failure and crop quality reduction. A study
reported that the weight and size of muskmelon fruits were improved
with the cadmium concentration of 50 and 200 mg/kg, but the soluble
sugar content decreased by 35.94% and 29.73% respectively, and the
cadmium content was far beyond the specified standard (Wang et al.,
2022). Therefore, cadmium treatment may improve crop yield, but it
does not guarantee crop nutrition and safety. Based on the above, the
research of cadmium pollution in agricultural soils urgently need to be
conducted.
Many research groups have proposed various solutions to the cad­
mium stress problem of different crops (Majumdar et al., 2020, J et al.,
2020, Hao et al., 2021). Biochar has become the excellent material for
the treatment of cadmium pollution in agricultural soil due to its good

* Corresponding author.
E-mail address: lijunli0424@sina.com (J. Li).
1
Co-first authors.

https://doi.org/10.1016/j.plaphy.2023.107661
Received 17 January 2023; Received in revised form 17 March 2023; Accepted 21 March 2023
Available online 21 March 2023
0981-9428/© 2023 Elsevier Masson SAS. All rights reserved.
Y. Cheng et al.
adsorption and fixation effect of cadmium (Chen et al., 2018; Zhang
et al., 2020b; Zhou et al., 2023). Biochar is a kind of carbon-rich material
formed by heating organic matter (such as straw, plant leaves, animal
manure, etc.) in an anaerobic or hypoxic environment(Li et al., 2023).
Biochar has many advantages ondealing with cadmium contamination
in agricultural soils. Biochar has the ability to promote crop absorption
of mineral elements and increase crop yields (Cao et al., 2021; Shin
et al., 2021). Moreover, biochar has a very low production cost and can
be prepared from agricultural wastes such as wheat straw, sawdust and
rice husk (Kumar et al., 2021). In recent years, biochar and its com­
posites have been studied for the ability of soil cadmium pollution
regulation. It is found that they have a good performance on the alle­
viation of cadmium stress for major crops such as rice, wheat and maize
(Abdul et al., 2021; Islam et al., 2021; Zhifan et al., 2021). Whether
biochar can generally alleviate cadmium stress in different crops re­
mains to be studied.
With the development and progress of science and technology,
transcriptome sequencing of biological samples has become simpler,
faster and less costly. With the ability of providing comprehensive gene
expression information of the organisms to be measured, transcriptome
sequencing has become a common way to study the molecular mecha­
nisms of biological systems (Li et al., 2021; Tian et al., 2021; Zhao et al.,
2021). This is exactly what is needed in the study of the mechanism of
crop resistance to stress. It has been widely used in the study of stress
such as cold (Mehmood et al., 2021), salt (Jiang et al., 2021), drought
(Waititu et al., 2021), and waterlogging (Yao, 2021). Transcriptome
analysis is also an effective strategy to study the molecular mechanism of
crop cadmium resistance and the mechanism of action of various crop
mitigation methods for cadmium stress mitigation. Enhancement of the
expression of genes related to diterpenoid biosynthesis and phenyl­
propane biosynthesis is of great benefit to rice roots against cadmium
stress (Wang et al., 2021b). When maize is under severe cadmium stress,
inoculation with arbuscular mycorrhizal fungi can promote the expres­
sion of antioxidant enzyme genes in maize and reduce the oxidative
stress damage to plants (Li et al., 2022). Therefore, the role of tran­
scriptome analysis in crop cadmium resistance research cannot be
ignored.
As an important cash crop, muskmelon has huge potential demand in
the world. At the same time, the safety of the muskmelon production is
also under the threat of cadmium pollution (Zhang et al., 2020a).
Therefore, physiological indexes detection and transcriptome
sequencing analysis were performed with muskmelon in this study, and
aims: (I) To evaluate the effect of biochar on alleviation of cadmium
stress in muskmelon and explore the mechanism of how biochar alle­
viates cadmium stress; (II) to study the gene expression strategy of
muskmelon in response to cadmium stress and the effect of biochar on
this strategy and to screen the key genes and metabolic pathways of
muskmelon in response to cadmium stress.
2. Materials and methods
2.1. Plant material and cultivation methods
The muskmelon seeds used in this study are sourced from the Hubei
Academy of Agricultural Sciences in China. The muskmelon was
cultured by soil culture, grouped and treated: free of cadmium and
biochar (CK), 400 mg/kg cadmium (T2), 400 mg/kg cadmium and 1%
biochar composite (T3), 400 mg/kg cadmium and 3% biochar composite
(T4), 400 mg/kg cadmium and 5% biochar composite (T5). 5 musk­
melon plants were planted per treatment group and 3 biological repli­
cates were set. The CdCl2⋅2.5H2O solid was configured as CdCl2 solution
for cadmium treatment of muskmelon. The biochar prepared from wood
was ground into particles, and the biochar powder obtained through the
40 mesh screen was used to control cadmium pollution. Mix CdCl2 so­
lution, biochar powder and 2500 g substrate nutrient soil in a culture
basin in a predetermined proportion and conduct aging treatment for
2
Plant Physiology and Biochemistry 197 (2023) 107661
two months. The muskmelon seeds are soaked in water at room tem­
perature for 3 h. Then wrap the muskmelon seeds in wet gauze, place it
in a thermostatic incubator, and germinate at 30 ◦ C for 24 h in the dark
conditions. The germinated seeds are sown in each hole in a nutrient
bowl filled with a matrix soil. When the muskmelon seedlings grow to
two euphylla stage, transplant it to the culture pots of each treatment
group to continue cultivating. During the second period, continuous
watering and fertilization were carried out to keep the soil moisture
content at 60–65%, and the main fertilizer was sulfur-containing tem­
porary compound fertilizer (N: P2O5: K2O = 15: 15: 15). Muskmelon
were sampled and tested after 40 days of culture.
2.2. Detection of cadmium content and physiological data
2.2.1. Measurement of growth data and collection and processing of
samples
The plant height of muskmelon and the length and width of the
leaves of eighth node were measured with a ruler. The hypocotyl
diameter of muskmelon was measured with a vernier caliper. The rela­
tive chlorophyll content of the leaves was measured with chlorophyll
meter SPAD-502 plus.
The leaves of eighth node and fruits of muskmelon were collected
(the fruit grew for about 40 days from pollination time, and the color of
the peel has turned from green to yellow). The leaves of each treatment
group were washed, dried and cut with scissors and mixed. The fruit skin
and seeds were removed and the fruit flesh was cut into small pieces. The
pulp and part of the leaves were dried in the oven for the detection of
cadmium content, and the remaining leaves were used for biochemical
indicators detection in the same way as in our previous article(Cheng
et al., 2020).
2.2.2. Detection of biochemical indicators
0.05 g leaves were weighed and extracted with ethanol-hydrochloric
acid solution (1%: 99%), and the absorbance of the extraction solution at
530 nm, 620 nm and 650 nm was measured with a spectrophotometer.
0.3g leaves were weighed and ground into homogenate at low temper­
ature by adding phosphate buffer (PB) with pH 7.8. The homogenate
was centrifuged at 4000 rpm/min for 10 min, and then the supernatant
was separated and diluted to 10 mL as the solution to be tested. 0.1 mL of
the tested solution, 0.9 mL PB with pH 7.8 and 5 mL of Coomassie Bright
Blue G250 solution were mixed, and stood in the dark for 5 min. The
absorbance of the mixture at 595 nm was measured to detect the content
of soluble protein; 2 mL of solution to be tested and 2 mL of 0.6% 2-thi­
obarbituric acid solution (Dissolved in 10% trichloroacetic acid solu­
tion) were mixed and placed in a boiling water bath for 15 min, and
centrifuged at 4000 rpm/min for 10 min, and the supernatant was taken
out and the absorbance at 532 nm, 600 nm and 450 nm was measured to
detect the amount of malondialdehyde (MDA) content; 0.5 mL of the
solution to be tested, 0.5 mL 130 mmol/L of methionine solution, 750
μmol/L nitrogen blue tetrazole solution, 100 μmol/L ethylenediamine
teodium solution, 20 μmol/L riboflavin solution and 3.5 mL PB with pH
7.8 was mixed and irradiated for 20min under 4000 lx fluorescent lamp
for 20 min, and the absorbance at 560 nm was measured to detect su­
peroxide dismutase (SOD) activity; 0.1 mL of the solution to be tested,
0.9 ml 100 mmol/L of PB with pH 6.0 and 3 mL of reaction solution (28
μL guaiacol and 19 μL 30% H2O2 were added to every 50 mL of 100
mmol/L PB with pH 6.0) were mixed and the absorbance at 470 nm was
measured every 30 s for 270 s to detect peroxidase (POD) activity; 0.1
mL of the solution to be tested, 0.3 mL of 30% H2O2 solution and 3.6 mL
PB were mixed, and the absorbance at 240 nm was measured every 10 s
for 90 s to detect the activity of catalase (CAT); 0.1 mL of the solution to
be tested, 20 μL 30% H2O2 solution, 5 μL ascorbate solution and 2.4 mL
phosphate buffer (0.1 mmL/L disodium ethylenediamine tetraacetic
acid was added to PB with pH 6.89) were mixed, and the absorbance at
290 nm was measure every 10 s for 90 s to detect the activity of
ascorbate peroxidase (APX).
Y. Cheng et al. Plant Physiology and Biochemistry 197 (2023) 107661

2.2.3. Cadmium content determination increased by 35.6% compared with CK group, while the content of MDA
0.1 g of dried muskmelon leaves and fruits were weighed and in T3, T4 and T5 groups decreased by 40.5%, 26.3% and 6.3% respec­
immersed in each test tube with 1 mL of concentrated nitric acid, and tively compared with T2 group. Usually, cadmium stress causes oxida­
were placed overnight. Then the test tube was placed in the boiling tive damage to plants and a decrease in plant biomass and chlorophyll
water bath for 1 h, taken out and cooled to normal temperature. 0.5 mL content. Correspondingly, plants will take a series of countermeasures to
of 30% H2O2 was added and the test tube is placed in the boiling water resist cadmium stress, including the accumulation of soluble proteins
bath for 30 min. The content of cadmium in the digested muskmelon and anthocyanins and the enhancement of enzyme activity in antioxi­
sample in the test tube was measured by atomic spectrophotometer. dant systems (Zhou et al., 2020; Wu et al., 2021a). Cadmium-treated
muskmelons did not differ significantly from the control group in
2.3. RNA extraction with Illumina sequencing many physiological indicators such as leaf length and leaf width, while
the detection results of chlorophyll and MDA still showed cadmium
According to the instruction manual, total RNA was extracted from stress on muskmelons. The results of the MDA test also showed the effect
leaves with RNAprep Kit (bio teke, Beijing, China). The concentration of biochar on alleviation of muskmelon cadmium stress to some extent.
and purity of each RNA sample were measured with NanoDrop 2000
spectrophotometer (Thermo Scientific, Wilmington, De, USA) and the 3.2. Transcriptome sequencing comparison
cDNA library was constructed. Then, the transcriptome was sequenced
using the Hiseq4000 platform (Illumina, San Diego, CA, USA) to obtain After we tried to sequence by transcriptome and fastp quality con­
the original data. Through the data quality control of the original data, trol, 15 samples had a volume of 114.98 Gb, and the reads volume of a
high-quality pure data can be obtained. Then the pure data were single sample was between 46.60 M and 74.24 M, with an average
compared to the reference genome DHL92-v3.6.1 of muskmelon, and PE amount of 53.22 M, and the average percentage of Q30 bases was
reads was compared with the reference genome sequence by Hisat2 93.02%. High-quality sequences were compared to annotation genes by
software to obtain the alignment results in the ordered BAM format. Salmon, and the Mapping ratios ranged from 83.76% to 86.84%, with an
average of 84.94% (Table 1). The overall results show that both the
2.4. Statistical analysis quantity and quality of the sequencing data meet the conditions for
downstream analysis.
IBM SPSS (ver. 21.) was used for physiological data of muskmelon
plants. Through statistical analysis, the data differences of different 3.3. Screening of differentially expressed genes(DEGs)
groups were determined by the minimum significant difference (LSD)
test at the level of 0.05. The results were expressed in the form of “mean We obtained the DEGs between groups based on transcriptome data.
± standard deviation” and plotted with origin 2021. According to the In order to study the effect of cadmium treatment on muskmelon gene
transcriptome sequencing data of melon plants, the R language package expression and the effect of biochar on muskmelon under cadmium
DESeq2 was used to complete the gene differential expression analysis, treatment, we selected T2 and CK (CK-vs-T2), T3 and T2 (T2-vs-T3), T4
and the hypergeometric distribution Phyper in R language was used to
complete the GO enrichment analysis and KEGG enrichment analysis of Table 1
DEGs. Statistics of sequencing data.
Treatments Total reads(M) Total bases(Gb) Q30(%) Mapping ratio
3. Results
CK-1 48.1 6.88 91.1 83.8
CK-2 52.1 7.40 93.2 85.0
3.1. Physiological indicators of muskmelon plants CK-3 46.6 6.73 93.1 84.8
T2-1 48.3 7.00 93.4 86.8
In this study, the physiological data of muskmelon showed that the T2-2 74.2 10.7 93.5 85.2
T2-3 48.9 7.18 92.7 84.9
treatment of cadmium and biochar did not cause significant change in
T3-1 56.3 8.09 93.2 85.2
leaf length, leaf width, hypocotyl thickness and plant height of musk­ T3-2 56.0 8.08 92.9 84.8
melon (Fig. 1A). And there was no significant increase in soluble protein T3-3 52.0 7.45 92.9 85.7
content and anthocyanin content in cadmium-treated muskmelon T4-1 48.6 7.10 92.3 83.9
(Fig. S1), and the activity of SOD, POD, CAT and APX in muskmelon also T4-2 53.2 7.60 93.7 84.7
T4-3 62.3 8.97 92.9 84.3
did not increase significantly (Fig. S2). However, cadmium treatment
T5-1 46.9 6.73 93.6 84.8
apparently reduced the chlorophyll content of muskmelon (Fig. 1B). In T5-2 52.2 7.51 93.5 85.4
our daily observations of muskmelons, we also found that cadmium- T5-3 52.5 7.57 93.4 84.8
treated muskmelon leaves have turned yellow. MDA is the product of
Notes:Each treatment was repeated 3 times.
plant oxidative damage. The content of MDA in T2 treatment group

Fig. 1. Some physiological indicators of muskmelon plants in different treatment groups were: (A) leaf length, leaf width, hypocotyl thickness and plant height; (B)
chlorophyll content; (C) MDA content. Different lowercase letters indicate significant differences at p < 0.05. The same as below.

3
Y. Cheng et al.
and T2 (T2-vs-T4), T5 and T2 (T2-vs-T5) differential genes for further
analysis. Muskmelon primarily resists cadmium stress by upregulating
gene expression, while biochar addition primarily downregulates gene
expression compared to cadmium treatment alone (Table 2). We then
performed GO enrichment and KEGG enrichment analysis on these DEGs
to study the function and effects of these genes on muskmelon.
3.4. GO function enrichment analysis
GO database annotations can be divided into three categories: Mo­
lecular function (MF), biological process (BP), and cellular component
(CC). Among T2-vs-CK, T3-vs-T2, T4-vs-T2 and T5-vs-T2, 311, 89, 6 and
70 genes were annotated by GO, respectively.
As shown in Fig. 2, in terms of BP, the DEGs of T2-vs-CK, T2-vs-T3
and T2-vs-T5 are enriched. GO terms include “metabolic process”,
“cellular process”, “biological regulation”, “regulation of biological
process” and “single-organism process”. The DEGs of T2-vs-T4 are
enriched in “metabolic process”, “cellular process” and “biological
regulation”. In terms of CC, the DEGs of T2-vs-CK, T2-vs-T3 and T2-vs-
T5 are enriched in the terms “cell part”, “cell”, “membrane” and
“organelle”, and the DEGs of T2-vs-T4 are annotated in the terms
“membrane”, “cell part” and “cell”. In terms of MF, “binding”, “catalytic
activity”, “transporter activity” and “molecular function regulator” are
the main enriched terms of DEGs of T2-vs-CK, T2-vs-T3 and T2-vs-T5,
and the DEGs of T2-vs-T4 are annotated in the terms of “binding” and
“catalytic activity”. The enrichment results of DEGs of T2-vs-CK, T2-vs-
T3 and T2-vs-T5 in BP, MF and CC showed high consistency. There were
few DEGs of T2-vs-T4, but their distribution entries were consistent with
the enrichment results of T2-vs-CK, T2-vs-T3 and T2-vs-T5.
3.5. KEGG functional enrichment analysis
We performed a KEGG enrichment analysis to identify the
biochemical metabolic pathways in which DEGs are involved. The
analysis showed that a total of 126 of these DEGs received KEGG an­
notations, which are 106, 20, 2 and 20 genes for T2-vs-CK, T3-vs-T2, T4-
vs-T2, and T5-vs-T2, respectively.
As shown in Fig. 3, the ten metabolic pathways are significantly
enriched for DEGs in each groups. The most significantly enriched
pathway in T2-vs-CK is “Phenylpropanoid biosynthesis”, followed by
“phenylalanine metabolism”. “Glycine, serine, and threonine meta­
bolism” is the most significantly enriched in T2-vs-T3, followed by
“phenylalanine metabolism”. The DEGs of T2-vs-T4 are located in the
pathway of “photosynthesis-antenna proteins” and “Cutin, suberine and
wax biosynthesis” respectively. “Phenylpropanoid biosynthesis
pathway” is also the most significantly enriched among the differentially
expressed genes of T2-vs-T5, followed by “phenylalanine metabolism”.
“Phenylpropanoid biosynthesis pathway” is a metabolic pathway with
significant enrichment of DEGs in many groups, which is likely crucial
for muskmelon to resist cadmium stress.
Table 2
Number of DEGs between two groups.
groups Total number of Number of up- Number of down-
DEGs regulated DEGs regulated DEGs
CK-vs- 394 301 93
T2
T2-vs- 111 19 92
T3
T2-vs- 7 2 5 participate in response to abiotic stress (Wang et al., 2021a). In this
T4
T2-vs- 92 3 89
T5
4
Plant Physiology and Biochemistry 197 (2023) 107661
3.6. Analysis of the phenylpropanoid biosynthesis pathway
Phenylpropanoid biosynthesis pathway includes the synthesis pro­
cess of phenylalanine into lignin. In plants, phenylalanine can gradually
synthesize p-hydroxyphenyl lignin, guaiacyl lignin, 5-hydroxyguaiacyl
lignin and syringyl lignin through the catalysis of pal, 4CL, CCR and
other enzymes (Fig. 4). In Fig. 5, we can find that cadmium treatment
apparently affected the expression of genes related to phenylpropanoid
biosynthesis pathway in muskmelon plants. The expression of two genes
encoding 4CL, three genes encoding pod, one gene encoding CCR, one
gene encoding COMT and two genes encoding CCoAOMT were signifi­
cantly up-regulated. Moreover, the addition of 1% and 5% biochar
changed the effect of cadmium on the expression of genes related to this
pathway. Compared with cadmium treated muskmelon plants, 1% bio­
char down-regulated the expression of one gene encoding 4CL, one gene
encoding pod and one gene encoding pal. 5% biochar had a more
obvious effect, which downregulated two genes encoding 4CL, two
genes encoding pod, one gene encoding CCR and one gene encoding pal.
However, compared with cadmium treated muskmelon, the addition of
3% biochar did not cause significant changes in gene expression.
3.7. Key genetic analysis of muskmelons against cadmium stress
After compared the DEGs of CK-vs-T2, T2-vs-T3, T2-vs-T4 and T2-vs-
T5, we found that in addition to phenylpropanoid related genes, there
are other same genes or genes with the same function. As shown in
Fig. 6, these genes are up-regulated in CK-vs-T2, while all down­
regulated in T2-vs-T5, T2-vs-T4 and T2-vs-T5. This is consistent with the
status of differential genes related to phenylpropanoid pathway in our
previous paper. These genes are co-responding DEGs to cadmium stress
in each treatment group and may be the key genes affecting the resis­
tance of muskmelon to cadmium stress.
3.7.1. Code for cytochrome P450 (CYP)
Cytochrome P450 family proteins are the largest family of enzymes
in plant metabolism, whose members are involved in many important
metabolic pathways such as Phenylpropanoid biosynthesis and gibber­
ellin biosynthesis (Li and Wei, 2020). There are 10 DEGs encoding cy­
tochrome P450 family proteins. The genes MELO3C022045.2,
MELO3C015511.2 and MELO3C005607.2 encode unknown protein
subtype; the gene MELO3C011930.2 encodes CYP71B34-like;
MELO3C015388.2 and MELO3C016687.2 encode CYP72A219-like;
MELO3C007592.2 encodes CYP81E8-like; MELO3C026488.2 encodes
CYP82A3-like; MELO3C026486.2 and MELO3C026491.2 encode
CYP82D47. Cadmium upregulated the expression of 7 of these genes,
while the addition of 1%, 3%, 5% biochar downregulated the expression
of 3, 2, 3 genes, respectively.
3.7.2. Annexin
The annexins in plants constitute a polygenic family involved in
responding to various stresses. Specifically, it has been found in rice
overexpressed when subjected to salt stress and water stress (Mallick
et al., 2021). Cadmium caused the overexpression of MELO3C018648.2,
whereas 1% biochar decreased the expression of this gene. What’s more,
5% biochar decreased the expression of another annex protein gene
MELO3C012455.2.
3.7.3. WRKY TFs
When plants are exposed to stress conditions, transcription factors
(TFs) activate downstream gene expression, signal transduction and
adaptation networks by combining with specific cis acting elements.
Many TF families in plants, such as WRKY TFs, have been identified to
study, cadmium stress increased the expression of one WRKY gene,
while 1% and 5% biochar decreased the expression of two and one
WRKY genes, respectively.
Y. Cheng et al.
Fig. 2. Results of GO enrichment analysis of DEGs in each group.
3.7.4. Plant hormone-related genes
Plant hormone, an indispensable regulator of plant growth and
development, plays an important regulatory role in all stages of plant
growth and development (Wu et al., 2021b). Muskmelon in cadmium
treatment group upregulated the expression of MELO3C003761.2 and
MELO3C011218.2, which encode the Cytokinin riboside 5′ -mono­
phosphate phosphoribohydrolase and gibberellin 3-beta-dioxygenase
1-like, respectively. However, the addition of 1% and 5% biochar
further downregulated the expression of these two genes.
3.7.5. Other genes
The lectins, a ubiquitous glycan-binding protein in plants, has mul­
tiple functions in cellular signaling during plant growth and develop­
ment, such as response to biotic and abiotic stresses (Naithani et al.,
2021). MELO3C022430.2 and MELO3C022429.2 are two amaranth
lectin genes, whose expression was apparently increased under the effect
of cadmium treatment. All of the addition of three concentrations of
biochar has down-regulated the expression of the former. What’s more,
the addition of 1% and 5% biochar has down-regulated the expression of
the latter. Potassium is one of the important mineral nutrients of plants,
5
Plant Physiology and Biochemistry 197 (2023) 107661
accounting for 2–10% of the dry weight of plants. It is not only necessary
for plants to maintain normal physiological and biochemical processes
such as photosynthesis, osmotic pressure regulation, protein synthesis,
etc., but also participates in response to biological and abiotic stress. but
plants’ absorption and transport of potassium is very dependent on
potassium transport protein (Cai et al., 2021). Cadmium treatment
upregulated a potassium transporter gene in muskmelon, while adding
1% and 5% biochar downregulated the gene expression in muskmelon.
Similarly, the expression levels of some genes, which encode prote­
ase inhibitory proteins and trypsin inhibitors, were upregulated to
varying degrees due to cadmium treatment.but downregulated by the
addition of biochar. Plant protease inhibitory proteins and trypsin in­
hibitors have important functions of defendence against various pests,
some of which have been found to show antiviral activity (Kirar et al.,
2022).
4. Discussions
Cadmium stress usually has a more obvious effect on the growth state
of plants, which is manifested as plant dwarfing, leaf yellowing and leaf
Y. Cheng et al. Plant Physiology and Biochemistry 197 (2023) 107661

Fig. 3. Results of KEGG enrichment analysis of DEGs in each group.

Fig. 4. Phenylpropanoid biosynthesis pathway (partial).

area reduction (Jayanti et al., 2021). It is possible that due to the cad­
mium resistance of muskmelon and environmental factors, many phys­
iological indicators did not reflect the adverse effects of cadmium on
muskmelon growth. However, the detection results of chlorophyll con­
tent and MDA content are enough to prove that muskmelon is subject to
cadmium stress. Transcriptome data from muskmelons leaves provides
further information for this. Through transcriptome analysis, we found
that muskmelon plants upregulated the expression of many genes to
cope with cadmium stress. Muskmelon activates the phenylpropanoid
biosynthesis pathway by promoting key enzymes synthesis such as PAL,
4CL, and CCR, following lignin synthesized in large quantities in
muskmelon. Lignin is an important component of the cell wall, which is
an important barrier that prevents cadmium from entering plant cells
(Wang et al., 2021b). The ability of the cell wall to bind heavy metal ions
depends on the amount of negative charge carried by the cell wall
components. The increase of the lignin content in plants can effectively
improve the effect of cell wall binding cadmium ions (Zhu et al., 2022).
Cadmium treatment also activated the flavonoid pathway of musk­
melon. Various flavonoids metabolites such as flavones and anthocya­
nins coming from flavonoids biosynthesis pathway are important

6
Y. Cheng et al.
Fig. 5. Heat map of DEGs of phenylpropanoid biosynthesis pathway in each treatment group.
antioxidant substances in plants, wich can effectively reduce the
oxidative damage caused by cadmium stress on plants (Ephrem et al.,
2019). At the same time, to cope with cadmium stress, muskmelons
upregulated the gene expression of plants such as cytochrome P450
family protein genes, annexin genes and WRKY genes, which is involved
in dealing with biological and abiotic stress. Moreover, the difference in
the expression of these genes also shows that muskmelons are subjected
to cadmium stress at the molecular level. The differences in gene
expression reflects the difference in the degree of cadmium stress of
muskmelons to a certain extent (Ma et al., 2021). Though combining
physiological and transcriptome analysis, we can not only understand
the molecular mechanism of plant resistance to cadmium stress, but also
more comprehensively and accurately evaluate the degree of cadmium
stress of plants.
We compared the DEGs among different groups and tried to evaluate
the degree of cadmium stress and the role of biochar at the molecular
level. Then we found a very interesting phenomenon. The results of GO
7
Plant Physiology and Biochemistry 197 (2023) 107661
enrichment showed that the most significant GO terms of differential
expression gene enrichment between cadmium treatment and control
was the same as it between biochar-cadmium composite treatment and
cadmium treatment. These differentially expressed gene sets have high
consistency, which indicates that biochar is likely to affect the degree of
cadmium stress on muskmelon. Further screening and analysis of the co-
responding DEGs of each group showed that cadmium treatment
downregulated genes in muskmelon with additional biochar applica­
tion. This indicates that muskmelons with additional biochar are less
susceptible to cadmium stress than cadmium-only treatments, in which
muskmelons have a weaker response to cadmium stress. Biochar atten­
uated cadmium stress to muskmelon, which is consistent with our
physiological results. The oxygen-bearing functional groups such as
hydroxyl and carboxyl groups of biochar can exchange with heavy metal
ions to fix heavy metals, which can improve the pH of soil environment
to limit the migration of heavy metal ions (Mansoor et al., 2021). This
may be the main mechanism of biochar to alleviate cadmium stress in
Y. Cheng et al. Plant Physiology and Biochemistry 197 (2023) 107661

Fig. 6. Heat map of co-responding DEGs in each treatment group.

8
Y. Cheng et al.
muskmelon. 5% of biochar fixed most of the cadmium in the soil,
making the cadmium content of muskmelon much lower than that of Cd
treated group (Fig. S3), thus having the best effect of alleviating cad­
mium stress. In leaves, 3% of biochar had the highest cadmium content,
and 1% of biochar group had a higher cadmium content than pure
cadmium group (Fig. S3 A). Biochar has been reported to promote the
uptake of nitrogen, iron, zinc, and copper by plants, but cadmium
competes with these elements (Dadasoglu et al., 2022). It is likely to be
that 1% or 3% biochar is not enough to fix most of the cadmium in the
soil. Biochar promotes the absorption of nutrient elements as well as free
cadmium ions. The sufficient supply of nutrients brought by biochar
may also help muskmelon resist cadmium stress, so 1% and 3% biochar
still show their effects to alleviate cadmium stress. 1% biochar can
adsorb enough nutrients. So compared with 1% biochar treatment, 3%
biochar absorbing more cadmium did not improve the stress resistance
of muskmelon, following the worse effect of alleviating cadmium stress.
Biochar may also affect the transport of cadmium in muskmelon,
significantly reducing the cadmium content in muskmelon fruit, of
which the specific mechanism needs further study. In conclusion, a
concentration of 5% can fix most of the cadmium in the soil contami­
nated by 400 mg/kg to achieve a good remediation effect. 1% and 3%
biochar were not enough to fix so much cadmium and even promoted
the absorption of cadmium by muskmelon. The application of 1% bio­
char can promote the absorption of nutrient elements to promote growth
and alleviate cadmium stress, for whichwhile higher concentrations of
biochar will not bring better effects. Therefore, for the lower degree of
soil cadmium pollution, it’s best to apply with the concentration which
is enough for fixing the majority of the cadmium in the soil. For the high
degree of soil cadmium pollution, it may be a better choice to apply with
the concentration which is enough for absorption of the nutritional el­
ements of muskmelon.
The genes related to the phenylpropanoid biosynthesis pathway as
well as co-responding genes such as cytochrome P450 family protein
genes, annexin genes and WRKY transcription factor genes are likely to
be key genes against cadmium stress in melon. In addition to the phe­
nylpropanoid biosynthesis pathway related genes mentioned above,
which can resist cadmium stress by promoting lignin synthesis, other
genes have functions related to resistance to stress, particularly the cy­
tochrome P450 family protein gene. Some studies have found that heavy
metal chromium can also cause P450 family protein gene over­
expression (Yu et al., 2020). Specifically, a P450 family protein gene in
wheat cells, TaCYP81D5, can accelerate the clearance of reactive oxygen
species and give wheat salt resistance (Meng et al., 2020), while
knocking out the gene encoding cytochrome P450 monooxygenase in­
duces rice cells to accumulate reactive oxygen species and die (Yuan­
jiang et al., 2020). The P450 family protein genes of these muskmelons
may also have the function of scavenging reactive oxygen species. Genes
encoding annexin, which is ubiquitous in plants, are activated in
response to various abiotic stresses and plant hormones (He et al., 2020).
Some studies have shown that annexin can enhance the tolerance of
plants to various stress to protect plants (Mallick et al., 2021). Espe­
cially, one study found that the corn annexin genes ZmANN33 and
ZmANN35 encode cell membranes repair protein during seed germina­
tion (He et al., 2020; Zhang et al., 2018). Existing studies have
confirmed that some WRKY family genes can improve the salt tolerance
and drought tolerance of plants by promoting abscisic acid synthesis
(Wu et al., 2022; Sun et al., 2022). What’s more, some studies have
reported WRKY Genes can regulate the development of rice (He et al.,
2021). Cytokinin riboside 5′ -monophosphate phosphoribohydrolase is a
member of the plant LOG protein family that can convert inactive
cytokinin nucleotides into bioactive cytokinins (Naseem et al., 2018).
Gibberellin 3-beta-dioxygenase is one of the three key enzymes in
gibberellin biosynthesis (Giacomelli et al., 2017). Exogenous application
of cytokinin has been shown to reduce oxidative damage caused by
heavy metal lead stress (Piotrowska-niczyporuk et al., 2018). Similarly,
leaf surface sprayed gibberellin can enhance the plant’s antioxidant
9
Plant Physiology and Biochemistry 197 (2023) 107661
defense and inhibit plant absorption and transport of cadmium (Sadiq
et al., 2021; Chen et al., 2021). We found that these screened genes have
stress-resistance-related functions. On the one hand, this shows that our
screening method is effective and these genes are likely to be key genes
in plants to resist cadmium stress. On the other hand, in addition to the
phenylpropanoid biosynthesis pathway related genes, cytochrome
P450, WRKY transcription factor and annexin gene all have huge family
members and complex functions. their role in muskmelon anti-cadmium
stress and whether they are effective in the anti-cadmium stress of other
crops deserve further exploration.
5. Conclusion
In addition to the physiological differences in each treatment group,
the gene expression of muskmelon also indirectly reflects the cadmium
stress situation. Combining physiological data with transcriptomic data,
we obtained more comprehensive and accurate information about
muskmelon in response to cadmium stress. Both cadmium treatment and
biochar compound treatment suffered from cadmium stress, but the
addition of three concentrations of biochar alleviated the cadmium
stress to varying degrees, which means biochar has a good control effect
on muskmelon cadmium stress. Since the effect of biochar to promote
the absorption of mineral elements may enable plants to ingest more
cadmium, 5% and 3% biochar has the best and worst effect to alleviate
cadmium stress in muskmelon, respectively. Phenylpropanoid biosyn­
thesis pathway is a pathway activated in all cadmium containing
treatment groups of muskmelon, which is a key pathway to resist cad­
mium stress. Overexpression of genes encoding enzymes such as PAL,
4CL, CCR and POD is the key to activate the phenylpropanoid biosyn­
thesis pathway. The cytochrome P450 family genes, WRKY genes and
annexin genes are probably the key genes for cadmium stress resistance
in muskmelon. Their specific role in the process of cadmium stress and
their cadmium resistance effect in other crops deserves further study.
We plan to further verify the function of these genes through gene
overexpression and knockout to study the actual effect and mechanism
of action of these genes in crop cadmium stress resistance. In addition,
the absorption and transport of cadmium and nutrient elements in plants
under different concentrations of biochar treatment will be studiedto
explore the influence and the mechanism of biochar concentration on
the absorption of cadmium and nutrients in crops. Our work will further
promote the application of biochar in cadmium pollution control in
agricultural soils.
Author statement
Yuxuan Cheng: Investigation, Formal analysis, Writing-Original
Draft, Writing-Review & Editing.
Lingzhi Qiu: Investigation, Formal analysis, Writing-Review &
Editing.
Pingkai Shen: Investigation, Formal analysis.
Yunqiang Wang: Supervision, Conceptualization, Validation,
Resources.
Junli Li: Supervision, Conceptualization, Resources, Project admin­
istration, Funding acquisition, Writing - Review & Editing.
Zhaoyi Dai: Supervision, Conceptualization, Validation, Resources.
Meifang Qi: Supervision, Conceptualization, Validation.
Ying Zhou: Investigation, Formal analysis.
Zhengkang Zou: Investigation, Formal analysis.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Y. Cheng et al. Plant Physiology and Biochemistry 197 (2023) 107661

Data availability Li, r, wu, y, lou, x, et al., 2023. Porous biochar materials for sustainable water treatment:
synthesis, modification, and application. water 15 (3), 395-395.
Li, y, faiz, a, moshage, h, et al., 2021. Comparative transcriptome analysis of inner blood-
Data will be made available on request. retinal barrier and blood–brain barrier in rats. Sci. Rep. 11, 12151.
Li, y, wei, k, 2020. Comparative functional genomics analysis of cytochrome p450 gene
superfamily in wheat and maize. BMC Plant Biol. 20, 93.
Acknowledgements Lijuan, h, bruun, h h c, xiaosong, y, et al., 2021. Effects of sulfur application on cadmium
accumulation in brown rice under wheat-rice rotation. Environmental pollution
This work was supported by China Agriculture Research System of (barking, essex : 1987) 287, 117601.
Ma, y, liu, k, zhang, c, et al., 2021. Comparative root transcriptome analysis of two
MOF and MARA [Grant No. CARS-25]; the Fundamental Research Funds soybean cultivars with different cadmium sensitivities reveals the underlying
for the Central Universities [Grant No. 2019IB005, 2020IB029, tolerance mechanisms. Genome 65, 27–42.
2021IA005]; Hubei Provincial Natural Science Foundation of China Majumdar, s, sachdev, s, kundu, r, 2020. Salicylic acid mediated reduction in grain
cadmium accumulation and amelioration of toxicity in oryza sativa l. Cv bandana.
[Grant No.2022CFB380]. Ecotoxicol. Environ. Saf. 205, 111167.
Mallick, s r, jadhao, k r, rout, g r, 2021. Overexpression of annexin gene in rice (oryza
sativa l.) for salinity and water stress. In Vitro Cell. Dev. Biol. Plant 57, 86–101.
Appendix A. Supplementary data
Mansoor, s, kour, n, manhas, s, et al., 2021. Biochar as a tool for effective management of
drought and heavy metal toxicity. Chemosphere 271, 129458.
Supplementary data to this article can be found online at https://doi. Mehmood, s s, lu, g, luo, d, et al., 2021. Integrated analysis of transcriptomics and
org/10.1016/j.plaphy.2023.107661. proteomics provides insights into the molecular regulation of cold response in
brassica napus. Environ. Exp. Bot. 187, 104480.
Meng, w, jiarui, y, lumin, q, et al., 2020. Tacyp81d5, one member in a wheat cytochrome
References p450 gene cluster, confers salinity tolerance via reactive oxygen species scavenging.
Plant Biotechnol.J. 18, 791–804.
Naithani, s, komath, s s, nonomura, a, et al., 2021. Plant lectins and their many roles:
Abdul, m, abid, n, muhammad, r, et al., 2021. Effects of biochar, farm manure, and
carbohydrate-binding and beyond. J. Plant Physiol. 266, 153531.
pressmud on mineral nutrients and cadmium availability to wheat (triticum
Naseem, m, bencurova, e, dandekar, t, 2018. The cytokinin-activating log-family proteins
aestivum l.) in cd-contaminated soil. Physiol. Plantarum 173, 191–200.
are not lysine decarboxylases. Trends Biochem. Sci. 43, 232–236.
Cai, k, zeng, f, wang, j, et al., 2021. Identification and characterization of hak/kup/kt
Piotrowska-niczyporuk, a, bajguz, a, zambrzycka-szelewa, e, et al., 2018. Exogenously
potassium transporter gene family in barley and their expression under abiotic stress.
applied auxins and cytokinins ameliorate lead toxicity by inducing antioxidant
BMC Genom. 22, 317.
defence system in green alga acutodesmus obliquus. Plant Physiol. Biochem. 132,
Cao, d, lan, y, chen, w, et al., 2021. Successive applications of fertilizers blended with
535–546.
biochar in the soil improve the availability of phosphorus and productivity of maize
Sadiq, r, maqbool, n, hussain, m, et al., 2021. Boosting antioxidant defense mechanism of
(zea mays l.). Eur. J. Agron. 130, 126344.
mungbean with foliar application of gibberellic acid to alleviate cadmium toxicity.
Cengiz, k, muhammad, a, nasser, a m, et al., 2020. Responses of nitric oxide and
Plant Physiol. Rep. 26, 741–748.
hydrogen sulfide in regulating oxidative defence system in wheat plants grown
Shin, j, park, d, hong, s, et al., 2021. Influence of activated biochar pellet fertilizer
under cadmium stress. Physiol. Plantarum 168 (2), 345–360.
application on greenhouse gas emissions and carbon sequestration in rice (oryza
Chen, h, yang, r, zhang, x, et al., 2021. Foliar application of gibberellin inhibits the
sativa l.) Production. Environ. Pollut. 285, 117457.
cadmium uptake and xylem transport in lettuce (lactuca sativa l.). Sci. Hortic. 288,
Sun, k, yue, y, wen, d, et al., 2020. Effects of exogenous sulfur on maize (zea mays l.)
110410.
Growth and cd accumulation in cd-contaminated plastic shed soil. Environ. Monit.
Chen, p, wang, h-y, zheng, r-l, et al., 2018. Long-term effects of biochar on rice
Assess. 192, 651.
production and stabilisation of cadmium and arsenic levels in contaminated paddy
Sun, s, li, x, gao, s, et al., 2022. A novel wrky transcription factor from ipomoea trifida,
soils. Earth Environ. Sci. trans. Royal Soc.Edinburgh 109, 415–420.
itfwrky70, confers drought tolerance in sweet potato. Int. J. Mol. Sci. 23, 686.
Cheng, y, cheng, y, zheng, h, et al., 2020. Evaluation and comparison of the toxic effects
Tian, h, guo, y, ding, m, et al., 2021. Identification of genes related to stress affecting
of mgo nps, zno nps, α-fe2o3 nps, γ-fe2o3 nps, and fe3o4 nps on the remediation for
thymus immune function in a chicken stress model using transcriptome analysis.
cadmium-related effects in wheat seedlings. Water, Air, Soil Pollut. 231, 7120–7128.
Res. Vet. Sci. 138, 90–99.
Dadasoglu, e, ekinci, m, turan, m, et al., 2022. Ameliorative effects of biochar for
Waititu, j k, zhang, x, chen, t, et al., 2021. Transcriptome analysis of tolerant and
cadmium stress on bean (phaseolus vulgaris l.) Growth. Sustainability 14 (23),
susceptible maize genotypes reveals novel insights about the molecular mechanisms
15563-15563.
underlying drought responses in leaves. Int. J. Mol. Sci. 22 (13), 6980-6980.
Ephrem, h, michela, d a, paolo, d f, et al., 2019. Genome-wide association mapping of
Wang, d, chen, q, chen, w, et al., 2021a. A wrky transcription factor, ejwrky17, from
total antioxidant capacity, phenols, tannins, and flavonoids in a panel of sorghum
eriobotrya japonica enhances drought tolerance in transgenic arabidopsis. Int. J.
bicolor and s. Bicolor × s. Halepense populations using multi-locus models. PLoS
Mol. Sci. 22, 5593.
One 14 (12), e0225979.
Wang, k, yu, h, zhang, x, et al., 2021b. A transcriptomic view of cadmium retention in
Giacomelli, l, masuero, d, vrhovsek, u, et al., 2017. Gibberellin metabolism in grapevine
roots of cadmium-safe rice line (oryza sativa l.). J. Hazard Mater. 418, 126379.
during bloom and fruit-set. Acta Hortic. 1157, 277–282.
Wang, y, lei, z, ye, r, et al., 2022. Effects of cadmium on physiochemistry and bioactive
Hao, y, lv, r, ma, c, et al., 2021. Graphitic carbon nitride (g-c3n4) alleviates cadmium-
substances of muskmelon (cucumis melo l.). Molecules 27 (9), 2913-2913.
induced phytotoxicity to rice (oryza sativa l.). Environ. Sci. Pollut. Res. 28,
Wu, m, zhang, k, xu, y, et al., 2022. The moso bamboo wrky transcription factor,
21276–21284.
phewrky86, regulates drought tolerance in transgenic plants. Plant Physiol.
He, x, liao, l, xie, s, et al., 2020. Comprehensive analyses of the annexin (ann) gene
Biochem. 170, 180–191.
family in brassica rapa, brassica oleracea and brassica napus reveals their roles in
Wu, s, wang, y, zhang, j, et al., 2021a. Exogenous melatonin improves physiological
stress response. Sci. Rep. 10, 4295.
characteristics and promotes growth of strawberry seedlings under cadmium stress.
He, y, zhu, m, li, z, et al., 2021. Ipa1 negatively regulates early rice seedling development
Hortic. Plant J. 7, 13–22.
by interfering with starch metabolism via the ga and wrky pathways. Int. J. Mol. Sci.
Wu, w, du, k, kang, x, et al., 2021b. The diverse roles of cytokinins in regulating leaf
22, 6605.
development. Hortic. Res. 8, 118.
Islam, m s, magid, a s i a, chen, y, et al., 2021. Effect of calcium and iron-enriched
Yao, q, 2021. Crucial waterlogging-responsive genes and pathways revealed by
biochar on arsenic and cadmium accumulation from soil to rice paddy tissues. Sci.
comparative physiology and transcriptome in tropical and temperate maize (zea
Total Environ. 785, 147163.
mays l.) Inbred lines. J. Plant Biol. 64, 313–325.
J, D y, f, C w, z, L f, et al., 2020. Physiological responses of peanut seedlings to exposure
Yu, x-z, lu, c-j, tang, s, et al., 2020. Transcriptomic analysis of cytochrome p450 genes
to low or high cadmium concentration and the alleviating effect of exogenous nitric
and pathways involved in chromium toxicity in oryza sativa. Ecotoxicology 29,
oxide to high cadmium concentration stress. Plant Biosys. Int. J. Deal. Aspect Plant
503–513.
Biol. 154, 405–412.
Yuanjiang, c, youlin, p, qiang, z, et al., 2020. Disruption of early lesion leaf 1, encoding a
Jayanti, y, prasanna, k, anaytullah, s, 2021. Effect of cadmium induced stress on
cytochrome p450 monooxygenase, induces ros accumulation and cell death in rice.
morphological growth and yield attributes of rice (oryza sativa l.). Res. Crops 22,
Plant J. : Cell Mol. Biol. 105, 942–956.
246–250.
Zhang, f, li, s, yang, s, et al., 2018. Retraction note to: overexpression of a cotton annexin
Jiang, w, pan, r, buitrago, s, et al., 2021. Conservation and divergence of the tasos1 gene
gene, ghann1, enhances drought and salt stress tolerance in transgenic cotton. Plant
family in salt stress response in wheat (triticum aestivum l.). Physiol. Mol. Biol.
Mol. Biol. 98, 185.
Plants 27, 1245–1260.
Zhang, j, wang, p, xiao, q, 2020a. Cadmium (cd) chloride affects the nutrient uptake and
Kirar, m, singh, h, sehrawat, n, 2022. Virtual screening and molecular dynamics
cd-resistant bacterium reduces the adsorption of cd in muskmelon plants. Open
simulation study of plant protease inhibitors against sars-cov-2 envelope protein.
Chem. 18, 711–719.
Inform. Med. Unlocked 30, 100909.
Zhang, s, quan, l, zhu, y, et al., 2020b. Differential effects of three amendments on the
Kumar, p s, gayathri, r, rathi, b s, 2021. A review on adsorptive separation of toxic metals
immobilisation of cadmium and lead for triticum aestivum grown on polluted soil.
from aquatic system using biochar produced from agro-waste. Chemosphere 285,
Environ. Sci. Pollut. Res. 27, 40434–40442.
131438.
Zhang, s, wu, x, peng, j, et al., 2021. Study of the physiological dynamics of cadmium
Li, j, liu, r, zhang, c, et al., 2022. Selenium uptake and accumulation in winter wheat as
accumulation in two varieties of rice with different cadmium-accumulating
affected by level of phosphate application and arbuscular mycorrhizal fungi.
properties, 2021 J. Chem. 2021 (6), 2–12, 6238893.
J. Hazard Mater. 433, 128762.

10
Y. Cheng et al.
Zhao, l, zhu, h, li, b, et al., 2021. Transcriptomic analysis of the disease-resistance
response in mandarins induced by the biocontrol yeast, yarrowia lipolytica. Biol.
Control 163, 104607.
Zhifan, c, jincheng, p, zhangdong, w, et al., 2021. A novel maize biochar-based
compound fertilizer for immobilizing cadmium and improving soil quality and maize
growth. Environ. Pollut. 277, 116455.
Zhou, j, cheng, k, huang, g, et al., 2020. Effects of exogenous 3-indoleacetic acid and
cadmium stress on the physiological and biochemical characteristics of
cinnamomum camphora. Ecotoxicol. Environ. Saf. 191, 109998.
11
Plant Physiology and Biochemistry 197 (2023) 107661
Zhou, p, adeel, m, guo, m, et al., 2023. Characterisation of biochar produced from two
types of chestnut shells for use in remediation of cadmium- and lead-contaminated
soil. Crop Pasture Sci. 74, 147–156.
Zhu, y, qiu, w, he, x, et al., 2022. Integrative analysis of transcriptome and proteome
provides insights into adaptation to cadmium stress in sedum plumbizincicola.
Ecotoxicol. Environ. Saf. 230, 113149.
Zou, m, zhou, s, zhou, y, et al., 2021. Cadmium pollution of soil-rice ecosystems in rice
cultivation dominated regions in China: a review. Environ. Pollut. 280, 116965.