agronomy
Article
RNA-Seq Transcriptome Analysis of Potato with Differential
Tolerance to Bentazone Herbicide
Jing Guo 1 , Xiuli Song 2 , Shiqi Sun 1 , Baihui Shao 1 , Bo Tao 1, * and Lili Zhang 1






Citation: Guo, J.; Song, X.; Sun, S.;
Shao, B.; Tao, B.; Zhang, L. RNA-Seq
Transcriptome Analysis of Potato
with Differential Tolerance to
Bentazone Herbicide. Agronomy 2021,
11, 897. https://doi.org/10.3390/
agronomy11050897
Received: 20 March 2021
Accepted: 29 April 2021
Published: 2 May 2021
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4.0/).
Agronomy 2021, 11, 897. https://doi.org/10.3390/agronomy11050897
1 College of Agronomy, Northeast Agricultural University, Harbin 150030, China;
Guojing1577667@163.com (J.G.); ttnnjia@163.com (S.S.); guojing1577667@126.com (B.S.);
zhanglilizw@163.com (L.Z.)
2 College of Geographical Sciences, Lingnan Normal University, Zhanjiang 524048, China;
Songxiuli5251@163.com
* Correspondence: botaol@163.com; Tel.: +86-04-5155190990
Abstract: Potato (Solanum tuberosum), an important food crop worldwide, is threatened by broadleaf
weeds. Bentazone is an effective herbicide for controlling weeds; however, as a photosynthesis in-
hibitor, it can also affect potato plants. Therefore, screening potato seedlings for bentazone resistance
and determining the genes involved is essential. Herein, we selected potato varieties with tolerance
and sensitivity to bentazone. The photosynthetic rate of sensitive plants was notably affected by
bentazone application, whereas the tolerant plants showed a significantly higher photosynthetic
rate. We observed 95.7% bentazone degradation within 24 d after application in the tolerant plants.
Transcriptome sequencing revealed that the numbers of differentially expressed genes (DEGs) be-
tween the tolerant and sensitive potato seedlings were 2703 and 11,024 before and after bentazone
application, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis
revealed that the majority of DEGs were enriched in metabolic pathways, biosynthesis of secondary
metals, carbon metabolism, glutathione metabolism, and photosynthesis. Polyphenol oxidase (PPO),
flavonoid 30 ,50 -methyltransferase-like (AOMT3), ribulose bisphosphate carboxylase small chain C
(RBCS-C), and chalcone synthase 2 (CHS2) were identified as candidates contributing to bentazone
tolerance. These results provide a theoretical basis for selecting potato stress-resistant resources in
the future.
Keywords: potato; bentazone; transcriptome; KEGG; candidate gene
1. Introduction
Potato (Solanum tuberosum L.) is the most important non-grain food crop worldwide
and is central to maintaining global food security. Potato is cultivated for its underground
storage stems (tubers), which are rich in starch and nutrients [1]. As the fourth major
food crop worldwide, S. tuberosum provides an important source of high-quality starch in
addition to being a nutritious food staple. Thus, potato plays an important role in the daily
lives of many, contributing to personal livelihood and diet as well as the economy [2–4].
Broadleaf weeds pose a substantial threat to potato production, impacting crop yields
through competition for resources and resulting in economic losses [5].
Bentazone (C10 H12 N2 O3 S), which is an important component of chemical herbicides
used in potato fields, is applied to control broadleaf weeds such as Galium spp. [6]. Benta-
zone is a postemergence herbicide that is applied in early spring to early summer. Crop
plants can rapidly metabolize bentazone to 6-OH- and 8-OH-bentazone, whereas the target
weeds cannot; thus, bentazone only disrupts photosynthesis in weeds, inhibiting their
ability to fix carbon and causing them to die [7,8]. For bentazone to be an effective herbicide,
the crop plants must not be sensitive to bentazone. Bentazone can produce phytotoxic
effects in some potato crops, which limits its usefulness for controlling broadleaf weeds
in potato fields. Screening potato seedlings for bentazone resistance can effectively solve
https://www.mdpi.com/journal/agronomy
Agronomy 2021, 11, 897 2 of 16

this problem, allowing for further development of bentazone as an efficient herbicide for
potato fields.
At present, research on bentazone resistance and the associated genes is very limited.
Lundegardh [9] discovered that bentazone resistance in a strain of the unicellular green
alga Monoraphidium pussilum was the result of a modified thylakoid membrane as well
as a metabolic change. In 2007, Zhang et al. identified the CYP81A6 (Bel) gene in rice
(Oryza sativa), which encodes a P450 hydroxylase that detoxifies the herbicide by catalyzing
bentazone hydroxylation [10]. The CYP81A6 gene was then introduced to Arabidopsis
and tobacco; plants expressing the protein showed tolerance to bentazone [11]. Recently,
Tao et al. [5] screened potato tissues for bentazone resistance; however, the molecular
mechanism of resistance has not been elucidated.
Many studies have investigated gene expression to determine plant defense and stress
mechanisms [12,13]. The transcriptional regulation of gene expression has been recognized
as an important component of plant response, resulting in changes at the biochemical, cellu-
lar, and physiological levels [14,15]. Understanding the molecular mechanisms underlying
herbicide resistance could facilitate the development of resistant crops, reducing the impact
of weeds on production. In this study, photosynthesis and bentazone degradation were
measured in resistant potato materials. To elucidate candidate genes involved in tolerance
to bentazone, transcriptome sequencing was conducted to identify the differential gene
expression between bentazone-sensitive and -tolerant potato materials.

2. Materials and Methods

2.1. Plant Materials
Potato seedlings with different resistance levels to bentazone were preserved in the
Department of Pesticide Research of Northeast Agricultural University. Bentazone aqueous
solution (48%) was purchased from Xingnong Pharmaceutical Co., Zhangzhou, China.

2.2. Potato Seedling Resistance to Bentazone

When the potato plantlets reached the five-leaf stage, the stems and leaves of the
sensitive (4–0) and tolerant (4–19) varieties were treated with bentazone at concentrations
of 1296 g a.i./ha, 648 g a.i./ha, and 324 g a.i./ha, at three replicates per concentration, with
each replicate containing 50 seedlings. The varieties were examined for damage, and the
survival rate was investigated on the fourth day after treatment. The leaves were scored as
follows: −, no damage to the leaves; X, focal burned spots on the leaves were <2 cm2 ; XX,
focal burned spots on the leaves were 2–4 cm2 ; XXX, the whole leaves were burned.

2.3. Determination of Photosynthetic Rate in Bentazone-Treated Potato Seedlings

When potato seedlings grew to the five-leaf stage under natural conditions, benta-
zone was applied to the leaves and stems of sensitive and tolerant potato seedlings at
concentrations of 1296 g a.i./ha, 648 g a.i./ha, and 324 g a.i./ha. A photosynthetic assay
apparatus was used to determine the photosynthetic rate of potato leaves (sampling at
9:00–11:00 a.m.) 0–15 d after spraying (randomly measure three plants, three leaves per
plant, and obtain the average value).

2.4. Determination of Bentazone Residues in Bentazone-Treated Potato Seedlings

When potato seedlings grew to the five-leaf stage under natural conditions, the
stems and leaves of bentazone-sensitive and -tolerant potato seedlings were treated with
1296 g a.i./ha bentazone, and treatment without bentazone was used as the control. Six to
ten fresh samples were collected at each time point, and the collection amount was greater
than 10 g, which was repeated three times. The sampling times were 0, 2, 4, 6, 8, 10, 12, and
24 d after bentazone application, and the samples were stored at −80 ◦ C until analysis. To
measure the bentazone residue, 10 g of potato seedlings with different resistance levels
were weighed, ground, and suspended in 80 mL of methanol. After oscillating for 2 h,
the extracted solution was placed in a 50 mL of polypropylene plug centrifugal tube and
Agronomy 2021, 11, 897 3 of 16

concentrated to 5 mL using a rotary evaporator. The concentrated solution was then diluted
with 80 mL of acetonitrile and extracted with 30 mL of n-hexane twice. The acetonitrile
phase was rotationally evaporated to 5 mL. High-performance liquid chromatography was
used to determine bentazone content in the sample [16].

2.5. Pretranscriptome Sample Preparation

The potato seedlings of the sensitive and tolerant varieties were treated with bentazone
(1296 g a.i./ha) when they reached the five-leaf stage, with each treatment consisting of
three biological repeats and each repetition including 10 potato seedlings. Potato seedlings
were collected after 24 h bentazone treatment and untreated. Then, 3 g of potato leaf tissues
were collected from each repetition, frozen in liquid nitrogen, and then stored at −80 ◦ C.

2.6. Transcriptome Sequencing and Assembly

We collected leaf tissues from nontreated and treated, confirmed sensitive and tolerant
potato plants. The treatments were designated as nontreated (susceptible without treatment,
TS), nontreated tolerant (tolerant without treatment, NT), treated susceptible (TS), and
treated tolerant (TT) plants. Tissues were collected for RNA extraction 24 h after bentazone
application, with three biological replicates per treatment. The samples were then sent to
Genesky Biotechnologies Inc., Shanghai, China, for transcriptome sequencing. Total RNA
was extracted from the samples and purified using a TruSeq Stranded Total RNA Library
Prep Kit (Illumina, San Diego, CA, USA). Agarose gel electrophoresis and an Agilent
2100 Bioanalyzer were used to detect the degree of RNA degradation (required RNA sample
concentration ≥100 ng/µL, total >2 µg, and RNA integrity number ≥6.5). Subsequently,
poly (A) mRNA was enriched using oligo (dT) magnetic beads and fragmented using
a fragmentation buffer (Agilent Technologies, Santa Clara, CA, USA). RNA fragments
were reverse transcribed into cDNA and detected using the Agilent 2100 Bioanalyzer
(required concentration >5 ng/µL; fragment length 300–400 bp; Agilent Technologies,
Santa Clara, CA, USA). The final sequencing products were validated for size using an
Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and sequenced
using a 2 × 125-bp paired-end sequencing module on an Illumina HiSeq 2500 (Illumina,
San Diego, CA, USA). Raw sequence reads were assessed for quality using the FastQC
software package (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed
on 30 September 2020)) and preprocessed to remove sequence adapters and low-quality
bases using Trimmomatic software [17].

2.7. Differential Gene Expression

HISAT2 software (https://ccb.jhu.edu/software/hisat2/index.shtml (accessed on
30 September 2020)) [18] was used to compare the filtered clean reads with the reference
database annotations (SolTub 3.0). The results of HISAT2 alignment were used to sta-
tistically evaluate experimental database abnormalities using RNA_seQc (http://www.
broadinstitute.org/cancer/cga/rna-seqc (accessed on 2 October 2020)) [19]. The known
mRNA was quantified using Cufflinks analysis protocol (http://cole-trapnell-lab.github.
io/cufflinks/ (accessed on 2 October 2020)), and Cuffdiff software was used to analyze the
differential gene expression between the experimental group and the control group [20].
The variance between samples was visualized using principal component analysis (PCA);
M-A plots were generated for each comparison of samples. The criteria for differential
gene expression included | log2 (fold change) | ed | log2 was visualized using principal
component analysis p fold ch [21]. Gene expression was compared between nontreated
tolerant and nontreated sensitive (NT vs. NS), treated tolerant and treated sensitive (TT
vs. TS), treated tolerant and nontreated resistant (TT vs. NT), and treated sensitive and
nontreated sensitive (TS vs. NS).
Agronomy 2021, 11, 897 4 of 16

2.8. Validation of Expression Using Quantitative Real-Time Polymerase Chain Reaction

According to the differential gene expression of sensitive and resistant varieties before
and after treatment, four candidate genes (AOMT3, CHS2, RBCS-C, and PPO) were selected
for verification. Total RNA was extracted from the potato leaves using an RNA out kit
(Sangong, Shanghai, China), and cDNA synthesis was performed using PrimeScript™ II 1st
Strand cDNA Synthesis Kit (TaKaRa, Shanghai, China). The mRNA expression levels of the
seven genes were determined using SYBR Premix Ex Taq (TaKaRa) in ABI 7300 real-time
PCR system (Applied Biosystems, Foster City, CA, USA). Their expressions were measured
using quantitative real-time polymerase chain reaction (q-PCR) based on the sequence
of the gene EF1α (elongation factor) [22]. Three biological replicates were performed for
each experiment. The relative gene expression was determined using the threshold cycle
method, and the FCs were calculated using the 2−∆∆ng formula [23]. The primers of the
genes and the internal reference genes provided in Table S1 in Supplementary Materials
were synthesized by Sangong in Shanghai. Gene amplification was performed, and the
dissolution curve is shown in Table S2.

3. Results
3.1. Difference Analysis of Potato Seedling Resistance to Bentazone
The sensitive (4–0) and tolerant (4–19) potato varieties could grow well at a low
concentration of 324 g a.i./ha, and the survival rate was >90%. However, at a concentration
of 1296 g a.i./ha, the survival rate of tolerant varieties was 90%, and that of sensitive
varieties was only 12% (Table 1). At the highest concentration of 1296 g a.i./ha, the leaves
of the sensitive (S) seedlings were entirely burned, whereas the leaves of the tolerant (T)
seedlings were slightly browned (Figure 1a). In the 648 g a.i./ha treatment, the leaves
of the S seedlings were scorched, whereas those of the T seedlings were not damaged
(Figure 1b). In the 324 g a.i./ha treatment, the leaves of both S and T potato seedlings
showed no damage (Figure 1c) (Table 2).

Table 1. Survival rate of bentazone at different concentrations.

Treatment Concentration (g a.i./ha) Varieties Survival Rate (%)

4–0 12
1296
4–19 90
4–0 62
648
4–19 96
4–0 98
324
4–19 100

Table 2. Leaf condition of sensitive and resistant potato seedlings treated with bentazone.

Treatment Concentration (g a.i./ha)

Different Varieties
1296 648 324
4–0 XXX XX −
4–19 X − −
Note: −, no damage to the leaves; X, focal burned spots on the leaves are <2 cm2 ; XX, focal burned spots on the
leaves are between 2 and 4 cm2 ; XXX, entire leaves are burned.
Agronomy 2021, 11, x 5 of 16
Agronomy 2021, 11, 897 5 of 16

Figure 1. Representative images of the leaf condition of sensitive (4–0) and tolerant (4–19) potato seedlings treated with
bentazone
Figure at a concentration
1. Representative imagesofof(a)the
1296 g a.i./ha,
leaf (b) 648
condition g a.i./ha, and
of sensitive (4–0)(c)and
324 tolerant
g a.i./ha.(4–19) potato seedlings treated with
bentazone at a concentration of (a) 1296 g a.i./ha, (b) 648 g a.i./ha, and (c) 324 g a.i./ha.
3.2. Effects of Bentazone on the Photosynthetic Rate of Sensitive and Resistant Potato Seedlings
3.2. Effects
The of Bentazone
stem on the
and leaves Photosynthetic
of potato seedlings Rate
were of Sensitive
treated withand
1296Resistant
g a.i./ha Potato Seedlings
of bentazone.
A significant difference
The stem and leavesinofphotosynthetic ratewere
potato seedlings was observed between
treated with 1296the S-CK and
g a.i./ha T-CK
of bentazone.
potato seedlings (p ≤ 0.01). From 0 to 15 d, there was a significant difference
A significant difference in photosynthetic rate was observed between the S-CK and between theT-CK
T and S seedlings after bentazone treatment, and the photosynthetic rate decreased by
potato seedlings (p ≤ 0.01). From 0 to 15 d, there was a significant difference between the
6.7% and 79.4%, respectively. Significant differences were observed between S and S-CK
T and S seedlings after bentazone treatment, and the photosynthetic rate decreased by
(p ≤ 0.05) from day 1 and between T and T-CK from day 5 (p ≤ 0.05) (Figure 2).
6.7% and 79.4%, respectively. Significant differences were observed between S and S-CK
(p ≤ 0.05) from day 1 and between T and T-CK from day 5 (p ≤ 0.05) (Figure 2).
Agronomy 2021, 11, x 6 of
Agronomy 2021, 11, 897 6 of 16

30
P = 0.5028 P = 0.4436 P = 0.7261 P = 0.0002
P = 0.0000
25 P = 0.0000 P = 0.0008
P = 0.0000 P = 0.0000
P = 0.0000 P = 0.0000 P = 0.0360
P = 0.0000 P = 0.0000
P = 0.0000
Photosynthesis rate

20 P = 0.0000 P = 0.0000
(μmol co2 m-2 s-1)

P = 0.0000 P = 0.0000
P = 0.0000
15 P = 0.0000

T−CK P = 0.0000
10 T
S−CK
5 S P = 0.0000
P = 0.0000

0
0 1 3 5 7 9 12 15
Time (day)

0d 1d 3d 5d 7d 9d 12 d 15 d
T−CK 0.2309 0.3000 0.4726 0.3055 0.3055 0.2309 0.4933 0.4509
T 0.2000 0.2517 0.4000 0.6000 0.2309 0.2517 0.2082 0.2517
SD
S−CK 0.2887 0.2000 0.4163 0.2000 0.3215 0.1528 0.5508 0.1528
S 0.2000 0.2517 0.5033 0.3055 0.2082 0.2517 0.8083 0.3000
T−CK 0.0092 0.0117 0.0179 0.0110 0.0111 0.0087 0.0190 0.0187
T 0.0080 0.0099 0.0152 0.0233 0.0098 0.0107 0.0088 0.0107
CV
S−CK 0.0130 0.0089 0.0182 0.0085 0.0143 0.0068 0.0252 0.0075
S 0.0090 0.0116 0.0246 0.0164 0.0126 0.0188 0.1183 0.0652

Figure 2. Effect of 1296 g a.i./ha bentazone on the photosynthetic rate of sensitive (S) and tolerant (T) potato seedlings over
15 d. T-CK,
Figure nontreated
2. Effect of 1296T gpotato
a.i./haseedlings;
bentazoneT, treated
on the Tphotosynthetic
potato seedlings;
rateS-CK, nontreated
of sensitive (S)Sand
potato seedlings;
tolerant S, treated
(T) potato S
seedlings over
potato
15 seedlings.
d. T-CK, A significant
nontreated difference
T potato was obtained
seedlings; by T
T, treated Duncan’s test.
potato seedlings; S-CK, nontreated S potato seedlings; S, treated S
potato seedlings. A significant difference was obtained by Duncan’s test.
3.3. Bentazone Residues in Sensitive and Resistant Potato Seedlings After Treatment
3.3.We
Bentazone
examined Residues in Sensitive
the remaining and Resistant
bentazone residues Potato Seedlings
in sensitive After Treatment
and resistant seedlings
after treatment with the same concentration of bentazone to identify differences
We examined the remaining bentazone residues in sensitive and resistant in their seedlin
ability to metabolize bentazone. Bentazone was detected in S and T varieties on day 0 (2 h),
after treatment with the same concentration of bentazone to identify differences in the
but there was no significant difference between S and T varieties. There was a significant
ability to metabolize bentazone. Bentazone was detected in S and T varieties on day 0
difference in the residual amount of bentazone between S and T at 2 d. Bentazone was
h), but there
gradually was noover
metabolized significant
time in difference between
both varieties; S and
however, T varieties.
degradation There
was fasterwas
in a signi
the tolerant variety. At 24 d, the amounts of residual bentazone in the T and S seedlingsBentazo
cant difference in the residual amount of bentazone between S and T at 2 d.
was1.4
were gradually
µg/kg and metabolized over time inwith
13.6 µg/kg, respectively, bothdegradation
varieties; however, degradation
rates of 95.7% and 60.6%,was fast
in the tolerant
respectively variety.
(Figure 3). At 24 d, the amounts of residual bentazone in the T and S seedlin
were 1.4 µ g/kg and 13.6 µ g/kg, respectively, with degradation rates of 95.7% and 60.6
respectively (Figure 3).
Agronomy 2021, 11, x 7 of 16
Agronomy 2021, 11, 897 7 of 16

40

35
S

Bentazon residue (μg/Kg)

30 T
P = 0.0004
25

20 P = 0.0000

15 P = 0.0000

P = 0.0000
10
P = 0.0000
5 P = 0.0000
P = 0.5039 P = 0.0000

0
0 2 4 6 8 10 12 24
Time (day)

0 d 2 d 4 d 6 d 8 d 10 d 12 d 24 d
S 0.1595 0.1528 0.1155 0.1000 0.1000 0.1528 0.1155 0.1000
SD
T 0.1405 0.1000 0.0000 0.0577 0.0577 0.0577 0.0577 0.0577
S 0.0302 0.0044 0.0039 0.0047 0.0056 0.0099 0.0080 0.0074
CV
T 0.0271 0.0030 0.0000 0.0030 0.0039 0.0046 0.0078 0.0102

Figure 3. Bentazone residues in the leaves of sensitive (S) and tolerant (T) potato seedlings treated
Figure 3. Bentazone
with 1296 residues A
g a.i./ha bentazone. in significant
the leavesdifference
of sensitive
was(S) and tolerant
obtained (T) potato
by Duncan’s test. seedlings treated
with 1296 g a.i./ha bentazone. A significant difference was obtained by Duncan’s test.
3.4. Quality Identification of Potato Seedlings with Different Resistance Levels
The transcriptomes
3.4. Quality Identificationofoftolerant and sensitive
Potato Seedlings withpotato seedlings
Different were Levels
Resistance sequenced before
and after treatment with bentazone to detect differences in gene expression. As shown in
TableThe
S3,transcriptomes
the Q20 and Q30of tolerant
values (i.e., and sensitive potato
the proportion seedlings
of sequencing errorwere sequenced
rate of bases less before
and after treatment with bentazone to detect differences in gene expression.
than 1% and 0.1%, respectively) of each sample was greater than 90%, and the percentage As shown in
Table S3,reads
of clean the Q20
was and Q30
greater values
than 99%. (i.e.,
Thesethe proportion
results indicate of
thatsequencing error rate of
the genetic sequences of bases
each
less sample
than 1% had
andhigh accuracy
0.1%, and quality,
respectively) meeting
of each the requirements
sample was greaterfor follow-up
than tests.
90%, and the per-
centage of clean reads was greater than 99%. These results indicate that the genetic se-
3.5. Principal Component Analysis
quences of each sample had high accuracy and quality, meeting the requirements for fol-
The individual samples in each of the four groups (NS, TS, NT, and TT) were gathered
low-up tests.
together, showing that the individual differences among groups were small. In addition,
the main components of NT and TT were similar. However, the main components of the
3.5.
NSPrincipal
and TS wereComponent
different,Analysis
confirming that mRNA expression in non-resistant varieties was
The by
affected individual samples
the bentazone in each
treatment of the
(Figure 4). four groupsvarieties
The resistant (NS, TS,showed
NT, and TT)
little were gath-
change
in mRNA
ered expression
together, showingbefore
thatand
theafter treatment;
individual thus, we can
differences conclude
among that were
groups the resistant
small. In ad-
varieties
dition, thewere
mainnotcomponents
sensitive to bentazone.
of NT and TT were similar. However, the main components
of the NS and TS were different, confirming that mRNA expression in non-resistant vari-
eties was affected by the bentazone treatment (Figure 4). The resistant varieties showed
little change in mRNA expression before and after treatment; thus, we can conclude that
the resistant varieties were not sensitive to bentazone.
 Agronomy
Agronomy 2021,
2021, 11,11,
897x 8 of
8 of 16 16

Figure
Figure 4. 4.Principal
Principalcomponent
component analysis plot
plot of
ofsamples
samplesfrom
fromexperimental
experimental and control
and groups.
control TS,
groups.
treated susceptible plants; NS, nontreated susceptible plants; TT, treated tolerant plants; NT,
TS, treated susceptible plants; NS, nontreated susceptible plants; TT, treated tolerant plants; NT, non-
treated tolerant
nontreated plants.
tolerant plants.

3.6.
3.6.mRNA
mRNADifferential
Differential Expression
ExpressionAnalysis
Analysis
InInthis
thisexperiment,
experiment,the thetype
typestandard
standardwas wasused
usedtotoscreen
screendifferentially
differentially expressed
expressed
genes
genes(DEGs), satisfyingp-values
(DEGs),satisfying p-values< <0.05
0.05andand| |log2
log2(fold
(foldchange)
change) | |> >1 1forfor
differential
differential
genes,
genes,where
wherelog2 log2(fold
(foldchange)
change)> >1 1is islabeled
labeledasasanan upregulated
upregulated gene
gene (Up);
(Up);log2
log2(fold
(fold
change) < − 1 is labeled as a downregulated gene (Down). There
change) < −1 is labeled as a downregulated gene (Down). There were more significant were more significant
differences
differences ininthethesusceptible
susceptible varieties
varietiesbefore
beforeandandafter treatment;
after treatment; a total
a total ofof10,661
10,661 DEGs
DEGs
were detected, among which 5329 were upregulated, and 5332
were detected, among which 5329 were upregulated, and 5332 were downregulated. were downregulated. In theIn
resistant varieties,
the resistant there were
varieties, therefewer
were differential genes before
fewer differential genesand after
before treatment;
and 1963 DEGs
after treatment; 1963
were detected, including 1097 upregulated genes and 866 downregulated.
DEGs were detected, including 1097 upregulated genes and 866 downregulated. Compar- Comparing
the
ingresistant and susceptible
the resistant varieties
and susceptible before
varieties treatment,
before therethere
treatment, werewere
2703 2703
DEGsDEGs in total, of
in total,
which 1696 were upregulated, and 1007 were downregulated; however,
of which 1696 were upregulated, and 1007 were downregulated; however, comparing the comparing the two
varieties after treatment,
two varieties a total ofa11,024
after treatment, total of DEGs wereDEGs
11,024 detected,
were5766 upregulated
detected, genes and
5766 upregulated
5258 downregulated genes (Table 3; Figure 5). There were 923 (539
genes and 5258 downregulated genes (Table 3; Figure 5). There were 923 (539 + 384) + 384) DEGs among
DEGs
TTamong
vs. NT, TT vs. TS, TS vs. NS (Figure 6). In summary, fewer significant
TT vs. NT, TT vs. TS, TS vs. NS (Figure 6). In summary, fewer significant differ- differences
between the resistant
ences between and susceptible
the resistant varietiesvarieties
and susceptible were present before treatment
were present than after
before treatment than
treatment, the gene expression of the susceptible variety showed significant changes after
after treatment, the gene expression of the susceptible variety showed significant changes
treatment, and the resistant variety had relatively fewer changes in gene expression after
after treatment, and the resistant variety had relatively fewer changes in gene expression
treatment. Thus, bentazone does not have a significant effect on the gene expression of the
after treatment. Thus, bentazone does not have a significant effect on the gene expression
resistant variety.
of the resistant variety.
 Agronomy2021,
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Figure5.5.Differential
Figure Differentialgene
geneexpression
expressionM-AM-Amaps.
maps.The
Theabscissa
abscissaisisthe
theAAvalue
valuelog2
log2(FPKM),
(FPKM),which
whichisisthe
thelogarithm
logarithmofofthe
the
expressionlevel,
expression level,representing
representingthe
thelevel
level
ofof gene
gene expression;
expression; thethe ordinate
ordinate is the
is the MM value
value log2
log2 (FC),
(FC), which
which is the
is the logarithm
logarithm of
of the ratio of the expression of the sample or group, used to measure the expression difference. The green dots
the ratio of the expression of the sample or group, used to measure the expression difference. The green dots represent the represent
the downregulated
downregulated differentially
differentially expressed
expressed genes (DEGs),
genes (DEGs), the red the
dotsred dots represent
represent upregulated
upregulated DEGs, and DEGs, anddots
the blue the represent
blue dots
represent genes that are not differentially expressed. NT, nontreated tolerant plants; NS, nontreated susceptible
genes that are not differentially expressed. NT, nontreated tolerant plants; NS, nontreated susceptible plants; TS, treated plants;
TS, treated susceptible plants; TT, treated tolerant plants.
susceptible plants; TT, treated tolerant plants.
 Agronomy 2021, 11, x 10 of 16

Agronomy 2021, 11, 897 10 of 16

Figure 6. Venn diagram: the number of differentially expressed genes common or specific to treated
Figure 6. Venn diagram: the number of differentially expressed genes common or specific to
and nontreated
treated tolerant
and nontreated andand
tolerant susceptible
susceptibleplants. NT,nontreated
plants. NT, nontreated tolerant
tolerant plants;
plants; NS, nontreated
NS, non-
treated susceptible
susceptible plants;plants; TS, treated
TS, treated susceptible
susceptible plants;
plants; TT,TT, treatedtolerant
treated tolerant plants.
plants.

Table 3. Differentially expressed genes putatively involved in differential tolerance to bentazone in potato.
Table 3. Differentially expressed genes putatively involved in differential tolerance to bentazone in potato.
Number of Differentially Expressed Genes
Number of Differentially Expressed Genes
Level
Level of Gene
of Gene Expression NT vs. NS TS vs. NS TT vs. NT TT vs. TS
NT vs. NS Up TS vs. NS Up
Down Down TTUpvs. NTDown Up TT vs. TS
Down
Expression
>1–2 Up Down 1251 Up 757 Down
2700 2651 Up 916 Down
648 2594Up 2590 Down
>1–2 >2–31251 757 315 2700 176 1184
2651 1364 916 129 136
648 14902594 1184 2590
>2–3 >3–4 315 176 83 1184 47 1364
600 695 129 35 13640 8441490 620 1184
>3–4 83 47 600 695 35 40 844 620
>4–5
>4–5 17 15
17 331 15 331
321
321 12 12 12
12 455455 309 309
>5 >5 3 5 3 359 5 359
176 176 1 1 99 258258 386 386
±inf ±inf 27 7 27 155 7 125
155 125 4 4 2121 125125 169 169
1696 1007 1696 53291007 5332
5329 5332 1097 1097 866
866 57665766 5258 5258
Total
Total 2703 10,661 1963 11,024
2703 10,661 1963 11,024
NTNTvs. vs.
NS:NS:
nontreated tolerant
nontreated relative
tolerant to nontreated
relative susceptible
to nontreated plants;
susceptible TS vs.TS
plants; NS:
vs.treated susceptible
NS: treated relativerelative
susceptible to nontreated
to non-susceptible
plants; TT vs. NT: treated tolerant relative to nontreated tolerant plants; TT vs. TS: treated tolerant relative to treated susceptible plants.
treated susceptible plants; TT vs. NT: treated tolerant relative to nontreated tolerant plants; TT vs. TS: treated tolerant
relative to treated susceptible plants.
3.7. Pathway Enrichment Analysis
3.7. Pathway Enrichment Analysis
The Kyoto Encyclopedia of Genes and Genomes (KEGG) is widely used as a reference
The Kyoto
database Encyclopedia
of pathway of Genes
networks forand Genomesdatasets
large-scale (KEGG) is widely used
generated as a reference
using high-throughput
database of pathway networks for large-scale datasets generated using high-throughput
sequencing technology. In order to understand the resistance mechanism of the bentazone-
sequencing technology. In order to understand the resistance mechanism of the benta-
tolerant potato variety, the TT vs. NT unigenes were compared with KEGG (p ≤ 0.05),
zone-tolerant potato variety, the TT vs. NT unigenes were compared with KEGG (p ≤ 0.05),
and the corresponding pathways were elucidated. Among 1963 DEGs, 677 (34.49%) were
and the corresponding pathways were elucidated. Among 1963 DEGs, 677 (34.49%) were
assignedtotoKEGG
assigned KEGG pathways.
pathways. AmongAmong theunigenes,
the 677 677 unigenes, 203 (29.99%)
203 (29.99%) were assigned
were assigned to to
metabolic pathways, which was the largest group among the KEGG categories.
metabolic pathways, which was the largest group among the KEGG categories. The next The next
largestpathway
largest pathway group
group waswas
the the biosynthesis
biosynthesis of secondary
of secondary metabolites,
metabolites, which included
which included 149 149
(22.01%) unigenes. The remaining pathway groups included carbon metabolism (36, 5.32%),
protein processing in the endoplasmic reticulum (31, 4.58%), glutathione metabolism
(17, 2.51%), carbon fixation in photosynthetic organisms (16, 2.36%), photosynthesis (14,
2.07%), and flavonoid biosynthesis (13, 1.92%) (Table S4).

3.8. Identification and q-PCR Validation of DEGs

Four candidate differential genes, polyphenol oxidase (PPO), flavonoid 30 ,50 -
methyltransferase-like (AOMT3), ribulose bisphosphate carboxylase small chain C
 (22.01%) unigenes. The remaining pathway groups included carbon metabolism (36,
5.32%), protein processing in the endoplasmic reticulum (31, 4.58%), glutathione metabo-
lism (17, 2.51%), carbon fixation in photosynthetic organisms (16, 2.36%), photosynthesis
Agronomy 2021, 11, 897 (14, 2.07%), and flavonoid biosynthesis (13, 1.92%) (Table S4). 11 of 16

3.8. Identification and q-PCR Validation of DEGs

Four candidate differential genes, polyphenol oxidase (PPO), flavonoid 3’,5’-methyl-
(RBCS-C), and chalcone synthase 2 (CHS2) were screened from the DEGs in resistant
transferase-like (AOMT3), ribulose bisphosphate carboxylase small chain C (RBCS-C), and
and sensitive plants based on2 the
chalcone synthase KEGG
(CHS2) werepathway
screened enrichment
from the DEGs analysis results.
in resistant These four
and sensitive plants
DEGs were based
significantly
on the KEGG pathway enrichment analysis results. These four DEGsvs.
downregulated in TS vs. NS and upregulated in TT TS.signifi-
were In
TT vs. NT, PPO was significantly downregulated, whereas AOMT3, RBCS-C,
cantly downregulated in TS vs. NS and upregulated in TT vs. TS. In TT vs. NT, PPO was and CHS2
were significantly upregulated.
significantly PPO expression
downregulated, whereas AOMT3, in resistant
RBCS-C,and andsensitive
CHS2 were varieties wasup-
significantly
significantly affected by bentazone treatment; the | log2 (fold change) | value of the by
regulated. PPO expression in resistant and sensitive varieties was significantly affected
PPO gene inbentazone
TS vs. NS treatment; the |higher
(7.59) was log2 (fold
than change)
that in| TT
valuevs.ofNT
the (1.78)
PPO gene in TS
group vs. NSS5).
(Table (7.59)
q-PCR results also showed that the AOMT3, RBCS-C, CHS2, and PPO genes in resistantthat
was higher than that in TT vs. NT (1.78) group (Table S5). q-PCR results also showed
the AOMT3,
potato seedlings RBCS-C, CHS2,
were significantly and PPO genes
upregulated afterinbentazone
resistant potato seedlings
application, were signifi-
showing a
cantly upregulated after bentazone application, showing a significant
significant difference with sensitive varieties (p ≤ 0.01), but PPO expression was downreg- difference with sen-
sitive varieties (p ≤ 0.01), but PPO expression was downregulated after bentazone appli-
ulated after bentazone application in both sensitive and tolerant varieties (Figure 7a). The
cation in both sensitive and tolerant varieties (Figure 7a). The expressions of AOMT3,
expressions of AOMT3, RBCS-C, CHS2, and PPO in tolerant and sensitive varieties after ben-
RBCS-C, CHS2, and PPO in tolerant and sensitive varieties after bentazone treatment were
tazone treatment were significantly
significantly higher
higher than those than
in the those in the
nontreated nontreated
varieties varieties
(p ≤ 0.01) (p ≤Although
(Figure 7b). 0.01)
(Figure 7b). PPO
Although PPO and
in sensitive in sensitive and resistant
resistant varieties varieties wasowing
was downregulated downregulated
to the actionowing
of benta-
to the actionzone,
of bentazone, PPO in
PPO expression expression in resistant
resistant varieties varieties was
was significantly significantly
higher than that inhigher
sensitive
than that in varieties.
sensitive varieties.

Agronomy 2021, 11, x 12 of 16

Figure 7. Expression analysis of the chosen transporters in tolerant and susceptible potato plants
before and
Figure 7. Expression after of
analysis bentazone
the chosentreatment. (a)inSensitive
transporters varieties
tolerant and treated
susceptible withplants
potato 1296 before
g a.i./ha
andofafter
bentazone
benta-
were
zone treatment. (a)compared with those
Sensitive varieties treatedthat were
with 1296untreated
g a.i./ha of(TS vs. NS).
bentazone The
were tolerantwith
compared varieties treated
those that were with
un-
treated (TS vs. NS).
1296 The tolerant
g a.i./ha varieties treated
of bentazone with 1296 with
were compared g a.i./ha of bentazone
those that were were compared
untreated withNT).
(TT vs. those
(b)that were
Tolerant
untreated (TT vs. NT). (b) Tolerant and sensitive varieties before bentazone treatment (NT vs. NS); tolerant and sensitive
and sensitive varieties before bentazone treatment (NT vs. NS); tolerant and sensitive varieties
varieties treated with bentazone (TT vs. TS). mRNA abundance was normalized using the housekeeping gene EF1α-actin,
treated
and the relative with bentazone
expression (TT vs. TS).
levels were calculated mRNA
using abundance
the 2−ΔΔCt was normalized
method. Three using were
biological replicates the housekeeping
performed. A
gene EF1α-actin, and the relative expression levels were calculated using the 2 −∆∆Ct method. Three
significant difference was obtained by Duncan’s test.
biological replicates were performed. A significant difference was obtained by Duncan’s test.
4. Discussion
4.1. Effects of Bentazone Application on Sensitive and Resistant Potato Varieties
Bentazone is a selective-contact herbicide that is widely used as a postemergence
treatment on soybean, wheat, and rice fields. Bentazone irreversibly blocks photosynthetic
electron transport in higher plants, inhibiting photosynthesis [24] and provoking oxida-
tive stress [25]. Bentazone is readily absorbed by leaves; however, the absorption and
translocation rate varies among plant species and varieties. In tolerant plants, absorption
Agronomy 2021, 11, 897 12 of 16

4. Discussion
4.1. Effects of Bentazone Application on Sensitive and Resistant Potato Varieties
Bentazone is a selective-contact herbicide that is widely used as a postemergence
treatment on soybean, wheat, and rice fields. Bentazone irreversibly blocks photosynthetic
electron transport in higher plants, inhibiting photosynthesis [24] and provoking oxidative
stress [25]. Bentazone is readily absorbed by leaves; however, the absorption and translo-
cation rate varies among plant species and varieties. In tolerant plants, absorption and
translocation of bentazone may be slower than in susceptible plants [26].
In this study, potato seedlings with different resistance levels were treated with
bentazone at concentrations of 1296 g a.i./ha, 648 g a.i./ha, and 324 g a.i./ha. As the con-
centration increased, leaves of the sensitive variety gradually curled or even died, while the
resistant variety showed little to no damage (Figure 1). The photosynthetic indices of T and
S potato seedlings were inhibited after treatment with bentazone (1296 g a.i./ha). However,
the inhibition effect was significantly higher in the S seedlings, and the photosynthetic
rate decreased dramatically over time (Figure 2). In the tolerant seedlings, the bentazone
in the leaves was 95.7% degraded at 24 d, which indicated that the tolerant variety could
rapidly metabolize bentazone (Figure 3). Plant tolerance to bentazone can be attributed to
increased metabolic breakdown [27].

4.2. DEGs in Bentazone-Resistant and -Susceptible Potato Seedlings

Functional genomics research using transcriptome sequencing technology is useful
for understanding the effects of gene expression regulation on potato gene functions and
the molecular mechanisms underlying important agronomic traits [1,28]. In this study,
transcriptome sequencing technology was used to examine gene expression in bentazone-
tolerant and -susceptible potato plants. Before bentazone application, 2703 DEGs were
detected among the two varieties. However, the number of DEGs significantly increased to
11,024 after bentazone application (Table 3).
In addition, several genes related to metabolism, photosynthesis, and anti-oxidation,
such as catalase, carboxylic oxidase, flavonoid transferase, polyphenol oxidase, and
glutathione-metabolizing enzyme, were affected by bentazone application. Zhu et al. [25]
reported that soybeans treated with bentazone exhibited a similar response. Many of the
bentazone-responsive genes identified are functionally categorized as protein families
involved in metabolism, stress response, and defense; the majority of these genes are
associated with abiotic stress response signaling and chemical detoxification pathways.
Numerous other herbicides and stress factors can induce stress response genes. In general,
abiotic stressors (such as herbicides, salinity, and drought) regulate the expression of genes
involved in signal cascades and transcriptional control [29]. These genes are activated to
counteract the effects of stress and maintain homeostasis. Metabolic enzymes, glutathione-
metabolizing genes, stress-signaling genes, detoxification-related genes, and antioxidants
were activated upon bentazone treatment in both sensitive and resistant potato plants
(Figure S1). Flavonoid transferase and polyphenol oxidase, for example, were upregulated
to counteract the oxidative stress resulting from lipid peroxidation after bentazone treat-
ment. This indicates that plants undergo extensive transcriptional adjustment in response
to herbicide-induced stress [30].

4.3. Screening of DEGs in Resistant Potato Plants

When comparing the DEGs among resistant and susceptible potato plants in this study,
PPO, AOMT3, RBCS-C, and CHS2 were significantly affected by bentazone application.
In plants, RBCS-C and PPO are important genes related to photosynthesis, and AOMT3
and CHS2 are important enzymes involved in flavonoid biosynthesis and metabolism.
PPO (polyphenol oxidase) is an enzyme with a dinuclear copper center that is mainly
associated with enzymatic browning [31]. It has also been shown to play an important
role in plant metabolism [32]. Additionally, PPO participates in the process of molecular
oxygen photoreduction in the plant photosynthetic system [33,34]). PPO also acts as a
Agronomy 2021, 11, 897 13 of 16

metal oxidoreductase to regulate the redox level in the cytoplasm, delivers molecular
oxygen to regulate photooxidation in the chloroplast, participates in electron transfer, and
plays a role in energy conversion [35]. The mechanism of bentazone by which it kills
weeds is the inhibition of photosystem II [36]. Research has shown that PPO is a plastid
enzyme present in the photosynthetic organelles (chloroplast thylakoid) of normal cells
and in non-photosynthetic plastids. PPO activity can be used to indicate the degree of
inhibition of photosynthesis and oxidative damage in plants. PPO protein is related to
photosystem II, and its activity is related to the high level of oxygen produced by chloro-
plasts, which indicates that PPO can prevent this inhibition by oxidizing the substrate
in plants. The oxygen and NADH required for PPO to catalyze phenolic substrates are
provided by the photosynthetic system, which plays a vital role in plant photosynthe-
sis, biosynthesis, resistance to external stress, and other physiological processes [37]. In
this study, the expression of PPO was significantly upregulated in TT compared to TS
(Figure 7b), indicating that the presence of the PPO gene in resistant plants under the
action of bentazone could weaken the effect of bentazone on photosynthesis, which is also
illustrated in Figure 2.
RBCS-C (ribulose bisphosphate carboxylase small chain C, chloroplastic) is a key
enzyme involved in photosynthesis, encoding ribulose carboxylase, which plays an impor-
tant role in the process of CO2 fixation [38]. Thus, RBCS-C can improve the CO2 fixation
capacity and photosynthetic efficiency of plants. Additionally, 42 RBCS genes were identi-
fied from the cDNA of Astragalus sinicus, which were shown to use C by fixing CO2 [39].
Frukh et al. [40] also reported that RBCS was involved in salt tolerance in rice. In this study,
the photosynthetic activity of susceptible plants was strongly inhibited after the applica-
tion of bentazone, whereas the tolerant plants were not significantly affected (Figure 1;
Figure 2). RNA-Seq and q-PCR results showed that the RBCS-C gene was significantly up-
regulated in the resistant plants compared to that in the S variety after bentazone application
(Figure 7b; Table S5). It was concluded that the RBCS-C gene is also one of the critical
reasons for bentazone resistance in resistant plants.
Flavonoids are polyphenols that contribute to plant growth, development, and stress
resistance; their biological functions have attracted much attention [41–43]. Flavonoids
are secondary plant metabolites, which have antioxidant and antistress characteristics.
They can improve cell function, activate the immune system, and protect against the
toxicity caused by pesticides [44]. Jhonsa et al. demonstrated that flavonoids could
reduce intracellular reactive oxygen species, regulate antioxidant defense systems, and
reduce paraquat toxicity [45]. AOMT3 (flavonoid 30 ,50 -methyltransferase-like) and CHS2
(chalcone synthase) are enzymes involved in flavonoid biosynthesis [46]. Among them,
CHS2 is the first enzyme in the flavonoid synthesis pathway as well as a key enzyme in
the secondary metabolism pathway of plants. The CHS2 gene also plays an important
role in Kochia scoparia resistance to the herbicide dicamba; increased CHS expression in
the meristem produces flavonols that compete with dicamba for intercellular transport by
ABCB transporters, resulting in reduced dicamba translocation [47]. In this study, AOMT3
and CHS2 were upregulated in T plants, which promoted the biosynthesis of flavonoids,
thus enhancing oxidative stress and subsequently increasing the tolerance to bentazone.
In conclusion, PPO, RBCS-C, AOMT3, and CHS2 play an important role in bentazone
resistance in plants. Our findings provide a preliminary framework for further physi-
ological and molecular study of bentazone in potato, which can be systematically and
comprehensively studied using a genomic approach in the future.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/

10.3390/agronomy11050897/s1, Table S1: Primers of q-PCR; Table S2: q-PCR gene dissolution
curve; Table S3: Sequencing data statistics; Table S4: NT vs. TT KEGG pathway analysis; Table S5:
Differentially expressed genes of four treatments (DEGs); Figure S1: NT vs. TT kegg_heatmap.
Agronomy 2021, 11, 897 14 of 16

Author Contributions: Methodology, B.T.; software, J.G.; validation, X.S., S.S. and B.S.; formal
analysis, J.G.; investigation, B.T. and L.Z.; resources, B.T.; data curation, J.G.; writing—original draft
preparation, J.G.; writing—review and editing, X.S. and J.G.; funding acquisition, B.T. and X.S. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Key Research and Development Plan in
China, the key technology and product development of chemical pesticide synergy, grant num-
ber 2016YFD0200503; Youth fund, regional joint fund of Guangdong basic and applied basic research
fund, grant number 2019A1515110888.
Institutional Review Board Statement: “Not applicable” for studies not involving humans or animals.
Informed Consent Statement: “Not applicable” for studies not involving humans.
Data Availability Statement: The data used to support the findings of this study are available from
the corresponding author upon request.
Acknowledgments: The authors wish to thank all the colleagues who assisted in this research and
provided technical advice and Xiuli Song for technical assistance.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.

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