LsMYB15 Regulates Bolting in Leaf Lettuce (Lactuca sativa L.) Under High-Temperature Stress

Abstract

High temperature is one of the primary environmental stress factors affecting the bolting of leaf lettuce. To determine the potential role of melatonin in regulating high-temperature induced bolting in leaf lettuce (Lactuca sativa L.), we conducted melatonin treatment of the bolting-sensitive cultivar “S39.” The results showed that 100 μmol L⁻¹ melatonin treatment significantly promoted growth, and melatonin treatment delayed high-temperature-induced bolting in lettuce. RNA-seq analysis revealed that the differentially expressed genes (DEGs) involved in “plant hormone signal transduction” and “phenylpropanoid biosynthesis” were significantly enriched during high-temperature and melatonin treatment. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis suggested that the expression patterns of abscisic acid (ABA)-related genes positively correlated with stem length during leaf lettuce development. Furthermore, weighted gene co-expression network analysis (WGCNA) demonstrated that MYB15 may play an important role in melatonin-induced resistance to high temperatures. Silencing the LsMYB15 gene in leaf lettuce resulted in early bolting, and exogenous melatonin delayed early bolting in leaf lettuce at high temperatures. Our study provides valuable data for future studies of leaf lettuce quality.

Full text

Frontiers in Plant Science | www.frontiersin.org 1 June 2022 | Volume 13 | Article 921021
ORIGINAL RESEARCH
published: 28 June 2022
doi: 10.3389/fpls.2022.921021
Edited by:
Peitao Lü,
Fujian Agriculture and Forestry
University, China
Reviewed by:
Tao Wu,
Hunan Agricultural University, China
Wang Huasen,
Zhejiang Agriculture and Forestry
University, China
*Correspondence:
Yingyan Han
hyybac@126.com
Specialty section:
This article was submitted to
Plant Systems and Synthetic Biology,
a section of the journal
Frontiers in Plant Science
Received: 15 April 2022
Accepted: 30 May 2022
Published: 28 June 2022
Citation:
Chen L, Xu M, Liu C, Hao J,
Fan S and Han Y (2022) LsMYB15
Regulates Bolting in Leaf Lettuce
(Lactuca sativa L.) Under High-
Temperature Stress.
Front. Plant Sci. 13:921021.
doi: 10.3389/fpls.2022.921021
LsMYB15 Regulates Bolting in Leaf
Lettuce (Lactuca sativa L.) Under
High-Temperature Stress
LiChen , MengnanXu , ChaojieLiu , JinghongHao , ShuangxiFan and YingyanHan *
Beijing Key Laboratory of New Technology in Agricultural Application, National Demonstration Center for Experimental Plant
Production Education, Beijing University of Agriculture, Beijing, China
High temperature is one of the primary environmental stress factors affecting the bolting
of leaf lettuce. To determine the potential role of melatonin in regulating high-temperature
induced bolting in leaf lettuce (Lactuca sativa L.), weconducted melatonin treatment of
the bolting-sensitive cultivar “S39.” The results showed that 100 μmol L−1 melatonin
treatment significantly promoted growth, and melatonin treatment delayed high-
temperature-induced bolting in lettuce. RNA-seq analysis revealed that the differentially
expressed genes (DEGs) involved in “plant hormone signal transduction” and
“phenylpropanoid biosynthesis” were signicantly enriched during high-temperature and
melatonin treatment. Quantitative reverse transcription polymerase chain reaction (qRT-
PCR) analysis suggested that the expression patterns of abscisic acid (ABA)-related genes
positively correlated with stem length during leaf lettuce development. Furthermore,
weighted gene co-expression network analysis (WGCNA) demonstrated that MYB15 may
play an important role in melatonin-induced resistance to high temperatures. Silencing
the LsMYB15 gene in leaf lettuce resulted in early bolting, and exogenous melatonin
delayed early bolting in leaf lettuce at high temperatures. Our study provides valuable
data for future studies of leaf lettuce quality.
Keywords: melatonin, leaf lettuce, high temperature, bolting, ABA-related gene
INTRODUCTION
Leaf lettuce (Lactuca sativa L.) is an annual vegetable crop of the Asteraceae family. It produces
a range of organic compounds, such as proteins, ber, phenols, and others, and has both
edible and medicinal value (Chadwick et al., 2016). As the cultivation of leaf lettuce expands
year on year and development continues, it has become one of the most-consumed leafy
vegetables in the world (Viacava et al., 2017). Leaf lettuce is primarily cultivated in polytunnels
and similar facilities and is prone to bolting (producing a owering stem prematurely) at high
temperatures during cultivation. Bolting is accelerated under high-temperature conditions, which
causes leaves to become bitter-tasting and limits crop marketability (Han et al., 2021a,b).
erefore, delayed bolting and owering are preferred in lettuce for maximizing harvestable
yield while maintaining culinary quality.
Leaf lettuce is a cruciferous vegetable and self-pollinating plant whose flowering is
accelerated under longer day lengths and higher ambient temperatures (Uwimana etal., 2012).

Frontiers in Plant Science | www.frontiersin.org 2 June 2022 | Volume 13 | Article 921021
Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
Premature flowering limits vegetative growth, and thus the
accumulation of sufficient resources for sustained growth.
Plants undergo profound physiological changes when
transitioning from vegetative to reproductive growth. Due
to its importance in both basic and translational biology,
flowering time has been extensively studied in model plant
species, particularly in Arabidopsis thaliana (thale cress; Han
et al., 2021a,b). Research has shown that, in Arabidopsis,
at least six genetic pathways regulate the transition to
flowering, including the photoperiod, vernalization,
autonomous, ambient temperature, age-dependent, and
gibberellin (GA) pathways, which coordinate with one another
to regulate flowering (Izawa, 2020). The signals from flowering
pathways, such as FLOWERING LOCUS T (FT), SUPPRESSOR
OF OVEREXPRESSION OF CO1 (SOC1), and LEAFY (LFY),
are integrated into the genetic network of flowering (Jutarou
et al., 2021). Among these integrons, the florigen gene FT
is the central node of floral transition, whose transcriptional
expression is positively regulated by zinc finger transcription
factor CONSTANS (CO), while negatively regulated by
FLOWERING LOCUS C (FLC; Zong et al., 2021). It has
been widely reported that different environmental factors
affect plant flowering by regulating the expression of floral
integrons and stimulating changes in plant hormone levels
(Zou etal., 2020). Increasing evidence suggests that flowering
time is associated with plant hormones (Ying et al., 2014).
In Arabidopsis, it has been found that the target of the
repressive function of abscisic acid (ABA) is the flowering-
promoting gene SOC1. One report has it that the
CORONATINE INSENSITIVE1 (COI1)-dependent signaling
pathway delays the flowering time by inhibiting the expression
of the florigen gene FLOWERING LOCUS T (FT). This
proved that the APETALA2 transcription factor (TFs) TARGET
OF EAT1 (TOE1) and TOE2 interact with JAZ (Jasmonate-Zim
domain) protein and repress the transcription of FT (Zhai
etal., 2015). Therefore, understanding the crosstalk between
hormones and flowering-related genes is of great importance
for elucidating the regulatory mechanisms of bolting and
flowering in plants.
In vegetable cultivation and production, leafy vegetables
that can bolt include, primarily, Chinese cabbage, beets,
spinach, celery, onion, and lettuce. The “low-temperature
vernalization” types of cruciferous vegetables have been
extensively studied. In Arabidopsis, FRIGIDA (FRI) and
FLOWERING LOCUS C (FLC) are important genes regulating
the vernalization pathway, and mechanistically interact to
control flowering time (Schon et al., 2021). FLC acts as a
strong repressor of floral transition; before vernalization,
expression of FLC is increased, and plants cannot bloom.
When plant vernalization is completed, expression of FLC
decreases, and the plants bloom. In Chinese cabbage (Brassica
rapa ssp. chinensis), BcFLC2 plays a key role in flowering
regulation as a negative regulator by controlling the expression
of BcSOC1, BcSPL15, BcMAF2, BcTEM1, and other genes
closely related to flowering (Huang et al., 2018). The
vernalization response in biennial beets (Beta vulgaris) is
mediated by the FT homolog BvFT1, which, in contrast to
the promotive action of FT in Arabidopsis, functions as a
repressor of flowering (Pin et al., 2010).
High temperature is one of the primary environmental stress
factors that aect the normal growth and development of plants
(Chen et al., 2017). Studies have found that environmental
temperature regulation of plant owering is aected by multiple
pathways. At ambient temperatures, FLC expression is
quantitatively modulated by a chromatin-silencing mechanism,
and the eect of chromatin on transcription and
co-transcriptional processing is of central importance to the
regulation of gene expression. e FT protein, acting as a
origen, is translocated from mature leaves to the shoot apex,
simultaneously forming a complex with FLOWERING LOCUS
D (FD). SOC1 is rst evoked in the shoot apex during the
oral switch process (Kong et al., 2016). e FT-FD dimer
and SOC1 activate the downstream genes LEAFY (LFY), AP1,
and FRUITIFUL (FUL) to dene oral characteristics. FLC
interacts with SHORT VEGETATIVE PHASE (SVP) to inhibit
SOC1 transcription by directly combining with the SOC1
promoter in the vegetative phase, among which SVP, FLM,
and their homologous genes play a key role (Balanzà et al.,
2014). SVP is a owering suppressor that inhibits plant owering
by combining with FT and SCO1 gene promoter elements.
e autonomous regulatory factors FCA and FVE participate
in the environmental temperature regulation pathway by
inhibiting SVP (Kong et al., 2016). In Arabidopsis, high
temperatures reduce SVP-FLC and promote owering (Jones
et al., 2020). SOC1 contributes to the GA-regulated oral
transition, and SOC1 expression is increased in response to
GA treatments. Studies have shown that the LsFT gene is
involved in the process of high temperatures promoting bolting
and owering in leaf lettuce, and using RNA interference
(RNAi) technology to knock down LsFT, the LsSOC1 gene
leads to delayed bolting and insensitivity to high temperatures
(Chen et al., 2017, 2018).
Melatonin (N-acetyl-5-methoxytryptamine) was rst
discovered in plants in 1995 (Kanwar etal., 2018), and whether
melatonin is universally present and what its function is in
plants has subsequently attracted widespread attention. e
presence of melatonin was reported in all plants tested and
in dierent parts of plants, including roots, stems, leaves,
owers, fruits, and seeds (Arnao and Hernández-Ruiz, 2020;
Onik et al., 2020), where the focus was on the eects of
exogenous melatonin on plant growth, morphogenesis, and
certain environmental stresses (Sun etal., 2020). Melatonin’s
actions include its ability to act as a plant biostimulator
against stress, both biotic and abiotic, its ability to regulate
plant growth, and its ability to regulate the processes of plant
vegetative development (Arnao and Hernández-Ruiz, 2018,
2019; Jahan etal., 2021a). In studies of senescence in kiwifruit
leaves, due to the enhanced scavenging activity of the antioxidant
enzymes peroxidase (POD), superoxide dismutase (SOD), and
catalase (CAT), membrane damage was reduced, and reduction
in the hydrogen peroxide (H2O2) content eectively delayed
senescence (Liang etal., 2018). In general, cold, heat, salinity,
drought, UV radiation, and chemical toxicity are countered
or mitigated by the presence of melatonin (Arnao and

Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
Frontiers in Plant Science | www.frontiersin.org 3 June 2022 | Volume 13 | Article 921021
Hernández-Ruiz, 2018; Choi and Back, 2019; Moustafa-Farag
et al., 2019). Melatonin promotes the relationship between
the growth of maize seedlings and the pathways related to
nitrogen metabolism, coordinating the growth and development
of maize seedlings and resulting in increased plant survival
rates, higher shoot and deeper root growth, and improved
photosynthetic eciency (Erdal, 2019). Melatonin also promotes
the relationship between grape seedling growth and sugar
metabolism, improving the resistance of grape seedlings,
accompanied by improved chloroplast and stomatal
morphologies (Zhong et al., 2020).
In this study, in order to obtain insights into transcriptional
regulation by melatonin treatment on early bolting and owering
in leaf lettuce under high-temperature stress, we used RNA
sequencing (RNA-seq) analysis to study exogenous melatonin
at high temperatures in the bolting-sensitive lettuce cultivar
“S39.” Transcriptome analysis revealed that plant hormone
signals, including ABA, ethylene, and GA signals, are involved
in this process. e level of LsMYB15, an ABA-related gene,
increases signicantly during this process. Based on an analysis
of the genes in the stem length-related gene module and using
weighted gene co-expression network analysis (WGCNA),
we determined that LsMYB15 plays an important regulatory
function in bolting in leaf lettuce. We propose that LsMYB15
could be a useful and eective genetic resource for the
improvement of leaf lettuce quality.
MATERIALS AND METHODS
Plant Materials and Growing Conditions
e bolting-sensitive L. sativa cultivar S39 was selected and
grown at the Beijing University of Agriculture Experimental
Station under greenhouse conditions [seeds provided by Cathay
Green Seeds (Beijing) Co., Ltd.]. e photoperiod was 16 h/8 h
(light/dark), the light intensity was maintained at
200 μmol m−2s−1, the temperature was maintained at 20°C/15°C
(day/night), and the relative humidity was 60–65%. Pest control
and water management were carried out according to standard
practices. A total of 300 uniform and disease-free plump seeds
were selected, which were then immersed in 9 cm-diameter
petri dishes lled with lter paper moistened with distilled
water. e dishes were then placed in a lighted incubator in
order to germinate. Following the development of a white
radical, the seeds were transplanted into a plug tray and placed
in a lighted incubator for cultivation.
Melatonin Treatment
When the leaf lettuce seedlings had reached the three-leaf-
center stage, they were transplanted to a nutrition bowl 11 cm
(diameter) × 12 cm (height), which remained in the light
incubator; plants displaying consistent growth conditions were
selected for testing. In October 2020, when the seedlings reached
the ve-leaf-center stage, they were subjected to a day/night
temperature setting of 20°C/15°C, a photoperiod of 16 h/8 h,
a light intensity of 200 μmol m−2s
−1, and a relative humidity
of 60–65%. Following initiation of the treatment, melatonin
(Sigma-Aldrich, Shanghai, China) was sprayed onto the plants
with a small sprayer at 9:00 a.m. on the morning of treatment
at concentrations of 0, 10, 50, 100, and 150 μmol L−1. Wearing
gloves, the spray was applied evenly to leaf surfaces until the
surfaces were wet but not dripping. Exogenous melatonin was
applied every 3 days, and following treatment for 15 days, the
plants were sampled at approximately 9:00 a.m., we selected
leaves that had been subjected to dierent concentrations of
melatonin in order to determine the physiological indicators
of the indices of the primary functional leaves.
In February 2021, when the seedlings had reached the
five-leaf-center stage, they were subjected to a day/night
temperature setting of 35°C/30°C, a photoperiod of 16 h/8 h,
a light intensity of 200 μmol m−2s−1, and a relative humidity
of 60–65% for high-temperature treatment. Following initiation
of the heat treatment, solutions of 0 μmol L−1 melatonin (H)
and 100 μmol L−1 melatonin (HM) were sprayed onto the
plants using the same small sprayer at 9:00 a.m. Again wearing
gloves, the spray was applied evenly to the leaf surfaces
until the surfaces were wet but not dripping. Exogenous
melatonin was applied every 3 days, and the plants were
subjected to high-temperature treatment for 30 days and
sampled at approximately 9:00 a.m. The leaf lettuce was then
immediately frozen in liquid nitrogen and stored at 80°C
for further analysis. We selected leaves (in triplicate) at 0,
6, 9, 15, 18, and 27 days that showed obvious changes in
phenotype, and the physiological indicators of the indices
of the primary functional leaves were determined and subjected
to RNA-seq analysis.
RNA Extraction and qRT-PCR Analysis
An RNA extraction kit (Aidlab, Beijing, China) was used to
extract total RNA from lettuce leaves according to the
manufacturer’s instructions, and RNA was converted into
complementary DNA (cDNA) using an Access RT-PCR system
(Promega Corporation, Madison, WI, UnitedStates). qRT-PCR
and SYBR Green qPCR Mix (Takara Bio Inc., Shiga, Japan)
and a Bio-Rad CFX96 real-time PCR system (Bio-Rad, Hercules,
CA, UnitedStates) were used to analyze gene expression levels.
e US National Center for Biotechnology Information (NCBI)
primer basic local alignment search tool (BLAST) algorithm1
was used to design PCR primers, which are listed in
Supplementary Table 3. Next, 10 μl 2 × SYBR Green qPCR
Mix (Takara Bio Inc.), 7 μl ddH2O, 1 μl upstream primer, 1 μl
downstream primer, and 1 μl 100 ng cDNA were added to tubes,
for a total volume of 20 μl. Aer mixing the reaction, the
program was set to 95°C for 30 s, 95°C for 10 s, 60°C for
15 s, and 72°C for 30 s for 39 cycles. Following completion of
the amplication cycle and cooling to 60°C, the DNA product
was denatured by heating it to 95°C, and a melting curve
was generated aer the operation was completed. e leaf
lettuce 18S ribosomal RNA gene was used as an internal control
to normalize transcript levels, and 2(−ΔΔCt) analysis was used
to calculate the transcript levels.
1
https://www.ncbi.nlm.nih.gov/tools/primer-blast

Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
Frontiers in Plant Science | www.frontiersin.org 4 June 2022 | Volume 13 | Article 921021
RNA-Seq Library Preparation and
Sequencing
RNA library preparation and sequencing were carried out. e
preliminary quality inspection included Nanodrop detection,
agarose gel electrophoresis detection, and Agilent 2100 detection,
which were used to determine RNA concentration, RNA integrity,
and RNA integrity number (RIN), respectively. A 1% agarose
gel was used to test for RNA degradation and contamination.
If the RNA bands were clear, it was taken that there were no
impurities or contamination. e RNA 6000 Nano kit was
used for testing, the kurtosis of the sample was concentrated,
and the RNA degradation rate was low, generally meeting the
requirements for standard library construction. According to
the recommendation of the manufacturer (Shanghai Majorbio
Biopharm Technology Co., Ltd), 33 sequencing libraries were
generated, the libraries were sequenced on an Illumina HiSeq
platform (Illumina, Inc., San Diego, CA, United States), and
paired-end reads were generated. SeqPrep soware2 and Sickle3
were used to perform quality control on the original sequencing
data to obtain high-quality quality control data (clean data)
to ensure the accuracy of subsequent analytic results.
Differential Expression Analysis
Aer obtaining the read counts of genes, the dierential
expression of genes between samples was analyzed to identify
dierentially expressed genes (DEGs) between samples, and
the functions of dierential genes were investigated. e
dierential expression module selects the DESeq2 soware to
analyze the raw counts. e default parameters are p < 0.05 &
|log2FC| > = 1; to control the probability or frequency of errors
in the overall inference result, the p value obtained by the
statistical test was subjected to multiple test corrections, which
is known as the p-adjust value. e platform uses BH (FDR
for correction with Benjamini/Hochberg) for multiple test
corrections of data. At values of p < 0.05, genes were considered
dierentially expressed.
GO and KEGG Cluster Analysis of
Differentially Expressed Genes
e gene ontology (GO) database4 was used to analyze genes
and gene products according to their participation in biological
processes (BPs), molecular functions (MFs), and cellular
components (CCs), which were classied and annotated according
to these three aspects. For p < 0.05, DEGs were considered
signicantly enriched. Kyoto Encyclopedia of Genes and Genomes
(KEGG)5 to perform KEGG pathway enrichment analysis on
the genes in the gene set. e calculation principle is the
same as that used in the GO function enrichment analysis.
Fisher’s exact test was used for enrichment analysis. e
Benjamini–Hochberg (BH) multiple calibration method was
chosen to check the p values. For corrected p < 0.05, the KEGG
2
https://github.com/jstjohn/SeqPrep
3
https://github.com/najoshi/sickle
4
http://www.geneontology.org
5
http://www.genome.jp/kegg
pathway that meets this condition was considered to be
signicantly enriched in the gene set.
Co-expression Analysis of Transcription
Factors and Phenotypic Data (WGCNA)
Weighted gene co-expression network analysis (WGCNA) was
used to identify modules of highly correlated genes based on
the FPKM data.6 Aer obtaining the gene modules that are
commonly expressed, welinked the modules to the phenotypic
information of interest to explore whether the expression of
transcription factors is related to high-temperature bolting genes.
Construction of and Infection With
LsMYB15 VIGS Vectors
e 340-bp open reading frames of LsMYB15 were used to
design the primers (Supplementary Table 3). e fragments
were then cloned from the S39 cDNA. e cloned fragments
and pTRV2 empty vector were digested with EcoR1 endonuclease
and BmaH1 endonuclease. Following purication, the fragments
were ligated and transformed to obtain a recombinant plasmid.
e identied recombinant plasmid was transformed into the
Agrobacterium strain GV3101, and an infection solution was
prepared. e infection buer consisted of 10 mM MgCl2,
10 mM MES, and 20 mM acetosyringone.
ree groups of plants were set up: a blank control group,
in which the plants received no injection (WT); a negative
control group, in which pTRV2 and pTRV1 empty vectors
were mixed in a 1:1 ratio and injected into the plants (TRV2);
and an experimental group, in which pTRV2-LsMYB15 and
pTRV1 empty vectors were mixed in a 1:1 ratio and injected
(TRV2-LsMYB15; Wang etal., 2021). Aer 1 week of infection,
the plants were held at 35°C for 1 week, and the other growth
conditions remained unchanged. Plant morphological changes
were monitored, including weekly measurements of stem length.
Aer 1 week of high-temperature treatment, the new leaves
that grew following the injection were randomly selected, then
tested using the characteristic tobacco rattle virus (TRV) primers
to determine whether the TRV virus had been transferred
into the plants, and nally treated with exogenous melatonin.
e cDNA of the young leaves was used to detect the eects
by PCR and qRT-PCR using gene-specic primers. To observe
morphological changes in the gene-silenced plants, the stem
lengths of the plants that were infected for 1 week were measured.
Enzyme-Linked Immunosorbent Assay
An Enzyme-Linked Immunosorbent Assay (ELISA) test kit
(ermo Fisher Technology Co., Ltd., Shanghai, China) was
used for testing in accordance with the manufacturer’s
instructions. e determination of antioxidant enzymes and
the extraction and determination of endogenous hormones
were both determined by ELISA. First, 0.1 g of lettuce leaves
was weighed and ground into a powder using liquid nitrogen.
e standard was then diluted, and samples were added according
6
https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/
Rpackages/WGCNA/

Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
Frontiers in Plant Science | www.frontiersin.org 5 June 2022 | Volume 13 | Article 921021
to the operating procedure and incubated at 37°C for 30 min.
Each well was lled with a washing solution, incubated for
30 s, and the washing solution then discarded, which was
repeated ve times. A 50-μl volume of enzyme-labeled reagent
was added to each well, except for blank wells, and incubated
at 37°C for 30 min. e washing was repeated ve times. Next,
50 μl of developer A and 50 μl of developer B were added to
each well, the plate was gently shaken, and the color was
developed at 37°C for 10 min in the dark. Finally, 50 μl of
stop solution was added to each well to stop the reaction (in
this case, the blue color turned yellow to indicate that the
reaction had stopped). Measurements were then taken within
15 min. e blank well was adjusted to zero, and the absorbance/
optical density (OD) of each well in sequence was measured
at a wavelength of 450 nm. e OD values of plant ascorbate
peroxidase (APX; EC 1.11.1.11), plant superoxide dismutase
(SOD; EC 1.15.1.1), plant catalase (CAT; EC 1.11.1.6), plant
peroxidase (POD; EC 1.11.1.7), phenylalanine ammonia lyase
(PAL; EC 4.3.1.5), and plant polyphenol oxidase (PPO; EC
1.10.3.1) were measured at 290, 560, 240, 450, 290, and 420 nm,
respectively. e ODs increased by 0.01, 0.01, 0.01, 0.01, 0.01,
and 0.001 units (U), respectively.
Data Analysis
Experimental data were analyzed using one-way ANOVA followed
by Tukey’s multiple range test to compare dierences between
the signicance of the dierence was p ≤ 0.05 or p ≤ 0.01. Origin
95, Microso Excel 2016 and IBM SPSS Statistics 22 were
used for analysis.
RESULTS
Effects of Treatment With Different
Concentrations of Melatonin on Growth
and Development in Leaf Lettuce
To examine the potential role of melatonin in regulating the
bolting-sensitive plant line S39, we treated plants with dierent
concentrations of exogenous melatonin (0, 10, 50, 100, and
150 μmol L−1), and then housed them under controlled-
temperature conditions (20°C) for 15 days (Figure 1A). e
results showed that exogenous melatonin signicantly altered
the blade length and blade width of leaf lettuce (Figure 1B).
Specically, a melatonin concentration of 100 μmol L−1 increased
the leaf length and leaf width of leaf lettuce by 45 and 52%,
respectively, compared to those treated with the lower
concentrations. erefore, weinferred that exogenous melatonin
aected the growth and development of leaf lettuce. e
preliminary experiment indicated that 100 μmol L−1 melatonin
signicantly enhanced growth vigor compared to the other
treatment groups, and the plants grew very well. However, a
melatonin concentration of 150 μmol L−1 resulted in serious
wilting. To verify whether melatonin also aected the growth
and development of leaf lettuce under high-temperature
conditions, weselected 100 μmol L−1 melatonin for subsequent
studies.
Transcriptome Analysis of Leaf Lettuce
Treated With Melatonin and Subjected to
High Temperatures
Leaf lettuce is susceptible to high-temperature stress during
cultivation, which causes bolting and owering, reducing the yield.
erefore, wetreated plants with 100 μmol L−1 melatonin to further
observe whether melatonin aected the growth of lettuce under
high-temperature conditions. Furthermore, wetreated leaf lettuce
with 100 μmol L−1 melatonin at high temperatures during the
growth period (the control group was not treated with exogenous
melatonin under the same conditions) and found that phenotypes
changed signicantly at 0, 6, 9, 15, 18, and 27 days. Compared
to the new phenotype, under the high-temperature treatment,
the control group without exogenous melatonin bolted signicantly
faster than the treatment group with exogenous melatonin. Moreover,
exogenous melatonin-treated lettuce leaves were larger and greener
than those in the control group (Figures 2A,B). erefore,
we selected the S39 cultivar for transcriptome analysis in order
to understand the exogenous melatonin regulation network of
bolting during leaf lettuce development at high temperatures.
Total RNA was extracted from three biological replicates of six
dierent leaf developmental stages of S39 and used to generate
cDNA libraries, which were subjected to paired-end sequencing
using Illumina high-throughput RNA sequencing. Aer removing
reads derived from rRNA and those of low quality, the total
length of clean reads ranged from 45,664,626 to 63,758,324 among
the dierent libraries, and almost 67% of the sequenced reads
could be aligned to the apple genome (Supplementary Table 1).
A Pearson correlation analysis indicated that the three libraries
from the biological replicates of each developmental stage had
highly consistent transcriptome proles (Supplementary Figure1).
Identication of Genes With Differential
Expression During Leaf Development
Under Melatonin Treatment
Fragments per kilobase of transcript per million mapped reads
(FPKM) values were used to investigate transcript dierences.
As a result, 399 genes (ratio > 2.0, p < 0.05) were found to
be upregulated and 506 genes (ratio < 0.5, p < 0.05) were
downregulated in HM6 vs. H6. e DEGs at this stage were
the lowest; 1,374 genes (ratio > 2.0, p < 0.05) were upregulated
and 968 genes (ratio < 0.5, p < 0.05) were downregulated in HM9
vs. H9; 1,352 genes (ratio > 2.0, p < 0.05) were upregulated and
1,034 genes (ratio < 0.5, p < 0.05) were downregulated in HM15
vs. H15 (Figures 2C,D); 2,182 genes (ratio > 2.0, p < 0.05) were
upregulated and 1,167 genes (ratio < 0.5, p < 0.05) were
downregulated in HM18 vs. H18. e expression level of
upregulated dierential genes was the highest, and 2,099 genes
(ratio > 2.0, p < 0.05) were upregulated and 2,960 genes (ratio < 0.5,
p < 0.05) were downregulated in HM27 vs. H27. e DEGs
were the most abundant at this stage (Figures2C,D). erefore,
we focused on the analysis of the HM27 vs. H27 stage with
the highest expression of DEGs in order to determine the
causes of leaf lettuce phenotypic changes.
DEGs (ratio > 2.0 or ratio < 0.5, p < 0.05) were classied into
dierent functional categories based on their GO annotations.

Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
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e numbers of DEGs involved in “metabolic processes” (MPs),
BPs, “catalytic activity” (CA), and MF were largest in HM27
vs. H27 (Figure 3A). In all the treatments, most DEGs were
from the BP and MF categories. KEGG is a pathway-related
database that provides a classication for biologically complex
patterns. Notably, KEGG terms associated with “plant hormone
signal transduction,” “starch and sucrose metabolism,” and
“phenylpropanoid biosynthesis” were enriched in HM27 vs.
H27 and in HM9 vs. H9, which are stages that are signicantly
aected by variations in bolting (Figure 3B). Genes involved
in “plant hormone signal transduction” were also enriched in
HM27 vs. H27 and in HM9 vs. H9, including several plant
hormone signal transduction and response proteins, including
ABA-related genes, auxin-responsive genes, ethylene signal
transduction pathway genes, and ethylene-responsive genes
(Supplementary Table 2). ese results suggested that, at high
temperatures, exogenous melatonin not only plays an important
role in leaf development but also aects bolting.
Identication of Important Modules and
Genes for Bolting Accumulation in Leaf
Lettuce Using WGCNA Analysis
Weighted gene co-expression network analysis (WGCNA) is
a method for identifying networks of genes with certain associated
functions or traits and for revealing putative hub genes with
particular inuence (Horvath et al., 2008; Yang et al., 2018).
To identify genes associated with bolting in leaf lettuce,
we identied co-expressed gene sets by applying WGCNA
(Figure 4A) to examine the genes expressed aer excluding
those with low FPKM values (average FPKM <1) and/or a
low coecient of variation (<1) across all development stages.
e 38,915 DEGs in the ve dierent treatment regimes that
met these stringent criteria were categorized into 35 co-expression
modules (Figure 4B).
Next, we measured the weight, height, blade length, blade
width, and steam length of leaf lettuce. e steam length of
leaf lettuce treated with melatonin at high temperatures was
shorter than that not treated with melatonin (Figures 4C,D).
An analysis of the relationship between modules and stem
length revealed that the Turquoise (r = 0.87, p = 3e−06) module
and the Blue module (r = 0.75, p = 3e−04) were highly correlated
with bolting (Figure 4B).
Genes associated with the “protein processing in endoplasmic
reticulum,” “ubiquitin-mediated proteolysis,” “photosynthesis-
antenna proteins,” “MAPK signaling pathway-plant,” and the
“phosphatidylinositol-signaling system” categories were enriched
in the Turquoise and Blue modules. Additionally, genes in the
“oxidative phosphorylation,” “glutathione metabolism,” “porphyrin
and chlorophyll metabolism,” and “carotenoid biosynthesis”
categories were also enriched in the Turquoise and Blue modules,
which are all processes that have been associated with leaf
lettuce bolting (Supplementary Figure 2).
To validate expression of the DEGs and bolting-related genes
from the Turquoise and Blue modules, qRT-PCR in leaf lettuce
was carried out for nine representative genes. e expression
levels of the bolting-related genes LsMYB15 (LG3315676,
XP_023766862.1), LsbHLH35 (LG5484648, PLY83907.1), LsZAT10
(LG198317, XP_023764915.1), LsNCED3 (LG7647715, PLY98266.1),
LsDPBF3 (LG8689275, XP_023768520.1), LsWNK6 (LG4344685,
XP_023729088.1), LsSIZ1 (LG1143132, XP_023733270.1), LsSPL12
(LG4402676, XP_023741755.1), and LsAGL27 (LG4402879,
A
B
FIGURE1 | Effects of different concentrations of melatonin treatment under control temperature (20°C) on growth and development in leaf lettuce. (A) Leaf lettuce
of the bolting-sensitive line S39 treated with 0, 10, 50, 100, and 150 μmol L−1 melatonin, corresponding to the morphological features of ve different concentrations.
Scale bars = 5 cm. (B) The weight, height, blade length, blade width, and steam length of leaf lettuce in ve different concentrations of melatonin. Different letters
above the bars indicate signicantly different values (p < 0.05) calculated using one-way analysis of variance (ANOVA) followed by a Tukey’s multiple range test.

Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
Frontiers in Plant Science | www.frontiersin.org 7 June 2022 | Volume 13 | Article 921021
XP_023741806.1) were examined. A correlation analysis showed
that the expression of these nine genes was closely related to
bolting (Figure5A). e DEGs between the control and melatonin
groups showed that, following melatonin treatment, signicant
upregulation of the expression of most regulatory genes in the
ABA-related genes occurred, except for the LsSPL12 gene
(Figure5B), as determined by RNA-seq. e relative gene expression
analysis of selected DEGs (LsMYB15, LG3315676) indicated high
integrity and correlation between the transcriptome analyses from
the RNA-seq and qRT-PCR results (Figure5C). is result indicated
that inhibiting bolting in leaf lettuce was positively aected by
melatonin treatment as a result of it increasing the expression
of ABA-related genes.
Silencing of LsMYB15 Expression in Leaf
Lettuce Alters the Occurrence of Bolting
Under drought or cold conditions, ABA is often recruited
as the primary signal for increasing the transcription levels
of stress-responsive genes that may confer protection to
assaulted plants (Agurla etal., 2018; Li et al., 2021). MYB
transcription factors have been found to play important
roles in many physiological processes under normal and
adverse growth conditions (Guo et al., 2017). MYB15 is a
member of the R2R3 MYB family of transcription factors
in Arabidopsis (Ding et al., 2009). However, the role of
MYB15 in high-temperature stress has not been previously
reported. In the work reported below, we provide evidence
A
B
CD
FIGURE2 | RNA-seq analysis of leaf lettuce treated with 100 μmol L−1 melatonin at high temperature during the growth period (the control group did not receive
exogenous melatonin under the same conditions). (A) In 100 μmol L−1 melatonin treatment (HM), the leaf developmental stages of phenotypes changed signicantly
at 0, 6, 9, 15, 18, and 27 days with different stem lengths, and whole plants were characterized. Scale bars = 5 cm. (B) In the control group that did not have
exogenous melatonin (H), the leaf developmental stages of phenotypes changed signicantly on 0, 6, 9, 15, 18, and 27 days with different stem lengths, and whole
plants were characterized. Scale bars = 5 cm. (C) Volcano plot visualizing differentially expressed genes (DEGs). The DEGs are shown in red and green. The x-axis
represents the fold change in HM6 vs. H6, HM96 vs. H9, HM15 vs. H15, HM18 vs. H18 and HM27 vs. H27 (on a log2 scale), and the y-axis represents the negative
log10-transformed p values (p < 0.05) of the t test for determining differences between the samples. (D) Cluster analysis of DEGs during different leaf treatments.

Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
Frontiers in Plant Science | www.frontiersin.org 8 June 2022 | Volume 13 | Article 921021
A
B
FIGURE3 | GO classication and KEGG pathway enrichment of differentially expressed genes (DEGs) in lettuce leaves. (A) GO classication of DEGs. (B) KEGG
pathway enrichment of DEGs. “MH” represents 100 μmol L−1 melatonin treatment under high temperature, and “H” represents no exogenous melatonin treatment
under high temperature.

Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
Frontiers in Plant Science | www.frontiersin.org 9 June 2022 | Volume 13 | Article 921021
showing that silencing of LsMYB15 leads to bolting in leaf
lettuce. To further elucidate the role of the LsMYB15 genes,
we suppressed their expression in leaves of the bolting-
sensitive S39 lettuce cultivar using virus-induced gene silencing
(VIGS) in the TRV vector.
e leaf lettuce plants inltrated with the virus containing
TRV2-LsMYB15 under high-temperature conditions and exposed
to exogenous melatonin administration, along with a control
group that did not receive exogenous melatonin or the viral
construct, developed a bolting phenotype 7 days post-infection.
In contrast, plants inltrated with the empty vector pTRV2
did not bolt (Supplementary Table 3). e rapid bolting in
plants expressing TRV2-LsMYB15 was then examined in the
control (Figures 6A,B). qRT-PCR revealed that the TRV2-
LsMYB15 transcript levels in the LsMYB15-silenced plants
decreased by approximately 90% compared to the control
plantlets. From the above phenotypic and physiological analyses,
the expression levels of ABA-related genes in LsMYB15-silenced
plants are of great signicance. For this purpose, qRT-PCR
was used to analyze and compare the gene transcription levels
between the LsMYB15-silenced plants and the control plants
(Supplementary Table 4). Based on the results from three
independent analyses, we found that the transcription levels
of three genes (LsMYB15, LsCOR15A, and LsRD29A) in the
LsMYB15-silenced plants were signicantly reduced in the
presence of exogenous melatonin compared to the melatonin-
treated control plants treated similarly. e transcript levels
of LsMYB15, LsCOR15A, and LsRD29A were also signicantly
lower in the LsMYB15-silenced plants than in the control group
in the absence of exogenous melatonin (Figure 6C). Taken
together, we further hypothesized that LsMYB15 plays a role
in transcriptional regulation via modulation of bolting and
indirect gene regulation, which could bethe cause of the altered
bolting in leaf lettuce.
A
C
D
B
FIGURE4 | Network analysis dendrogram showing modules identied by weighted gene co-expression network analysis (WGCNA). (A) Dendrogram plot with
color annotation. (B) Module-bolting correlations and corresponding p values (in parentheses). The left panel shows the seven modules. The color scale on the right
shows module trait correlation from 1 (green) to 1 (red). (C) The weight, height, blade length, blade width, and steam length of leaf lettuce in ve developmental
stages with exogenous melatonin at high temperature. “MH” represents 100 μmol L−1 melatonin treatment under high temperature. (D) The weight, height, blade
length, blade width, and steam length of leaf lettuce in ve developmental stages without exogenous melatonin at high temperature. “H” represents no exogenous
melatonin treatment under high temperature. Different letters above the bars indicate signicantly different values (p < 0.05) calculated using one-way analysis of
variance (ANOVA) followed by a Tukey’s multiple range test.

Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
Frontiers in Plant Science | www.frontiersin.org 10 June 2022 | Volume 13 | Article 921021
e results of antioxidant enzyme activity measurements
showed that APX, SOD, CAT, POD, and PAL activities exhibited
a steady increase in both the control plants and the
melatonin-treated leaf lettuce, with no signicant dierence
between WT and TRV2, but PPO activity decreased rapidly
(Figure 6D).
A
B
C
FIGURE5 | Identication and analysis of bolting-related genes. (A) Heatmaps describing the expression proles of candidate genes related to bolting-related
genes. (B) Validation of RNA-seq expression proles via qRT-PCR. (C) Correlation analysis between ABA-related genes and expression of the related candidate
LsMYB15 (LG3315676) via RNA-seq and qRT-PCR data. “MH” represents 100 μmol L−1 melatonin treatment under high temperature, and “H” represents no
exogenous melatonin treatment under high temperature. Different letters above the bars indicate signicantly different values (p < 0.05) calculated using one-way
analysis of variance (ANOVA) followed by Tukey’s multiple range test.

Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
Frontiers in Plant Science | www.frontiersin.org 11 June 2022 | Volume 13 | Article 921021
DISCUSSION
Effects of Melatonin on Leaf Lettuce Under
High-Temperature Stress
Melatonin is considered a potential plant growth regulator that
enhances plant growth. Nevertheless, the role of melatonin in
mediating the stress response in dierent plant species and
growth cycles still needs to beexplored. Melatonin can enhance
the tolerance of plants to abiotic stresses such as drought,
salt, ambient temperature, and heavy metals in dierent ways
(Wang et al., 2017a,b; Sun et al., 2020). For example, in rice
seedlings, exogenous melatonin treatment under salt stress
increased the net photosynthetic rate of rice and enhanced
the absorption and transmission of light energy, increasing
the relative water content and sucrose and starch contents and
improving the salt tolerance of rice (Yan etal., 2021). In wheat
(Triticum aestivum L.), the application of melatonin reduced
oxidative stress by lowering thiobarbituric acid-reactive substances
A
C
D
B
FIGURE6 | Silencing of LsMYB15 genes in the bolting-sensitive line S39. (A) Under high-temperature conditions, the exogenous melatonin administration
constructs developed a bolting phenotype 7 days postinfection. Scale bars = 5 cm. (B) Under high-temperature conditions, the control group that did not have
exogenous melatonin construct developed a bolting phenotype 7 days postinfection. Scale bars = 5 cm. (C) Relative expression levels in inoculated TRV2-LsMYB15
plants were determined using qRT-PCR to determine transcription levels of three genes (LsMYB15, LsCOR15A, and LsRD29A) in leaf lettuce plants. (D) Activities of
APX, SOD, CAT, POD, PAL, and PPO during heat treatment of control and melatonin-treated leaf lettuce. Bars indicate the standard error from each mean of three
independent replications. Mean values with the same letter are not signicantly different (p < 0.05). An increase in absorbance of 0.01 at 290 nm, 0.01 at 560 nm,
0.01 at 240 nm, 0.01 at 450 nm, 0.01 at 290 nm and 0.001 at 420 nm per min was dened as one unit (U) of APX, SOD, CAT, POD, PAL, and PPO activity,
respectively. “MH” represents 100 μmol L−1 melatonin treatment under high temperature, and “H” represents no exogenous melatonin treatment under high
temperature. Error bars indicate the SEM of three replicate measurements. Different letters above the bars indicate signicantly different values (p < 0.05) calculated
using one-way analysis of variance (ANOVA) followed by Tukey’s multiple range test.

Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
Frontiers in Plant Science | www.frontiersin.org 12 June 2022 | Volume 13 | Article 921021
and H2O2 content, increasing the activity of antioxidant enzymes
and of photosynthesis and carbohydrate metabolism and actively
improving heat resistance (Iqbal et al., 2021).
In conclusion, melatonin is extensively involved in the plant
stress response, regulating growth and development and
protecting plants from abiotic stress. High temperature is one
of the primary environmental stress factors that aect the
normal growth and development of leaf lettuce. Leaf lettuce
growth temperatures above 30°C result in early bolting, and
bolting reduces both plant quality and yields (Chen et al.,
2017). erefore, we selected 100 μmol L −1 melatonin at high
temperatures during the growth period (the control group was
not treated with exogenous melatonin under the same conditions)
for further research. We found that the growth of leaf lettuce
recovered following melatonin treatment at high temperatures,
while the speed of bolting was signicantly faster in the control
group than in the treatment group. In addition, exogenous
melatonin-treated lettuce leaves were larger and greener than
those in the control group, with the most signicant leaf width
being 49% higher than that of the control group (Figures2A–D).
ese results demonstrated that melatonin is involved in the
stress response of leaf lettuce, regulating its growth and
development and protecting leaf lettuce from high-temperature
stress.
Plant Hormones Play an Important Role in
Enhancing Plant Growth and Stress
Resistance
Phytohormones participate in various processes throughout a
plant’s lifecycle. Studies have shown that ve classical plant
hormones, including auxins, cytokinins, GAs, ABA, and ethylene,
play important roles in plant growth and stress responses, as
well as brassinosteroids, jasmonic acid, salicylic acid,
strigolactones, and peptides (Wang et al., 2017a,b; Zhao et al.,
2021). For example, in cucumber seedlings, indoleacetic acid—
acting as a downstream signaling molecule—is involved in
H2S-induced tolerance to chilling (Zhang et al., 2020). In
Arabidopsis, high temperature promotes ABA accumulation,
and elevated ABA levels trigger the upregulation of ABA enzyme
biosynthesis genes, improving plant heat tolerance during growth
(Gupta et al., 2020). In conclusion, hormones play a key role
in adaptation to abiotic stress during plant growth and
development. In this study, the number of DEGs involved in
“plant hormone signal transduction” was signicantly enhanced,
as determined by KEGG analysis (Supplementary Figure 2).
Using WGCNA, some of the genes screened from the high-
correlation modules, including MYB15, bHLH35, ZAT10, NCED3,
DPBF3, WNK6, SIZ1, and SPL12, were all ABA-related and
have been reported to regulate plant growth and stress tolerance
by regulating the ABA pathway (Figure5). In perennial ryegrass
(Lolium perenne) leaves, exogenous melatonin reduced the ABA
content, delaying senescence. e reduction in ABA was correlated
with downregulation of two ABA biosynthesis genes (LpZEP
and LpNCED1), which were upregulated by heat stress, although
melatonin suppressed this eect. us, the response of ABA
in heat-induced senescence was delayed by melatonin through
the reduction in ABA biosynthesis and the downregulation of
signaling pathway factors (Zhang et al., 2017). We believe,
therefore, that ABA plays an important role in the growth
and development of lettuce under high-temperature conditions.
Melatonin exerts its eects by regulating various elements
related to interfering with the activities of other phytohormones.
For example, studies have found that, following exogenous
melatonin treatment of tomato (Solanum lycopersicum) plants,
the expression levels of ABA receptors induced by melatonin
were increased and ABA signaling transduction pathways were
activated, leading to heat resistance and inhibition of heat-
induced senescence in tomato plants (Jahan et al., 2021b). In
cucumber (Cucumis sativus) seedlings, exogenous melatonin
were found to alleviate low-temperature stress by upregulating
the expression of CsZat12 and regulating the metabolism of
PA and ABA (Zhao et al., 2017). In conclusion, ABA plays
an important role in melatonin therapy and improves the
tolerance of plants to abiotic stress. e results of this study
demonstrate that melatonin regulates the growth and development
of leaf lettuce by regulating the expression of ABA-related
genes under high-temperature conditions, consistent with the
results of previous studies.
LsMYB15 Regulates Bolting in Leaf
Lettuce
e MYB transcription factor plays an important role in plant
physiological processes under conditions of treatment with
exogenous melatonin. In tea (Camellia sinensis) leaves, the
increase in lignin content was found to parallel the rise in
POD activity following melatonin treatment, which revealed
that melatonin could increase lignin accumulation by enhancing
POD activity. Additionally, melatonin also modied the expression
of MYB transcription factor genes, which were regarded as
candidates for the regulation of lignin metabolism in tea leaves
(Han etal., 2021a,b). In water bamboo shoots, the transcription
factors of ZlMYB1 and ZlMYB2 from the MYB family were
increased, and melatonin treatment markedly reduced their
expression, indicating that melatonin may be a participant in
the transcriptional regulation of lignin synthesis (Yang et al.,
2022). LsMYB15 (LG3315676) was selected from the identied
DEGs as a putative regulator of bolting using WGCNA. In
Arabidopsis, MYB15 is a positive regulator of drought and salt
tolerance. Enhanced transcript levels of ABA biosynthesis,
signaling, or responsive genes in MYB15 overexpression lines
also lead to increased tolerance to drought and salt stress
(Ding et al., 2009; Li et al., 2021). When induced by
low-temperature stresses, C-repeat binding factor (CBF) functions
as a positive regulator to aid ABA-dependent increases in the
expression levels of stress-responsive genes, leading to enhanced
tolerance to freezing (Zhou etal., 2011). ere were no changes
in COR15A or RD29A in MYB15-overexpressing plants,
suggesting that the coordination of upstream genes induces
downstream genes more eectively with cellular protective
functions (Chen etal., 2010). ese studies suggest that melatonin
treatment directly upregulates defense genes in response to
stress through the ABA pathway, decreasing cellular injury.

Chen et al. LsMYB15 Regulates Bolting in Leaf Lettuce
Frontiers in Plant Science | www.frontiersin.org 13 June 2022 | Volume 13 | Article 921021
In this study, LsMYB15 silencing resulted in decreased
expression levels of the downstream genes LsCOR15A and
LsRD29A. ese results indicate that MYB15 may function
dierently and beinvolved in separate transcriptional regulation
schemes under dierent stresses. From the results of the
phenotypic, physiological, and molecular analyses conducted
on leaf lettuce treated with exogenous melatonin under high-
temperature conditions, strong positive correlations were observed
among LsMYB15-silenced genes and reduced expression of the
genes involved in ABA signaling (Figure 6). Furthermore,
downstream stress–response pathways were activated, including
those for reduced antioxidant systems, reduced osmoprotectants,
enhanced membrane oxidation, and enhanced leaf senescence.
In the presence of melatonin, leaf lettuce also bolted.
Consequently, LsMYB15 is likely to be a positive regulator of
high-temperature bolting in leaf lettuce.
In conclusion, we have provided evidence that LsMYB15
regulates bolting in leaf lettuce. ese studies provide a more
detailed understanding of the high-temperature stress regulatory
network during leaf lettuce development and a new perspective
for studying bolting and the potential applications of hormone
treatment in agriculture.
DATA AVAILABILITY STATEMENT
e datasets presented in this study are publicly available, this
data can be found here: https://www.ncbi.nlm.nih.gov/bioproject/
PRJNA810911.
AUTHOR CONTRIBUTIONS
LC: data curation, formal analysis, methodology, and writing—
original dra. MX: soware, resources, and validation. CL:
project administration, resources, soware, and supervision.
JH: methodology and validation. SF: supervision, resources,
and funding acquisition. YH: conceptualization, writing—review
and editing, and project administration. All authors contributed
to the article and approved the submitted version.
FUNDING
Financial support was provided by e National Natural Science
Foundation of China (32172607) and the Beijing Municipal
Organization Department-Top-notch Young Talents Program
(2018000026833ZK76).
ACKNOWLEDGMENTS
We thank Cathay Green Seeds (Beijing) Co., Ltd. for providing
experimental resources.
SUPPLEMENTARY MATERIAL
e Supplementary Material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fpls.2022.921021/
full#supplementary-material
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