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Frontiers in Plant Science | www.frontiersin.org 1 August 2021 | Volume 12 | Article 715683
ORIGINAL RESEARCH
published: 11 August 2021
doi: 10.3389/fpls.2021.715683
Edited by:
Chris Helliwell,
Commonwealth Scientic and
Industrial Research Organisation
(CSIRO), Australia
Reviewed by:
Jingbo Zhang,
St. John's University, UnitedStates
Ana M. Casas,
Aula Dei Experimental Station (EEAD),
Spain
*Correspondence:
Zhenghong Li
luculia_gratissima@163.com
Youming Wan
wanyouming@126.com
Hong Ma
hortscience@163.com
Specialty section:
This article was submitted to
Plant Development and EvoDevo,
a section of the journal
Frontiers in Plant Science
Received: 27 May 2021
Accepted: 16 July 2021
Published: 11 August 2021
Citation:
Liu X, Wan Y, An J, Zhang X, Cao Y,
Li Z, Liu X and Ma H (2021)
Morphological, Physiological, and
Molecular Responses of Sweetly
Fragrant Luculia gratissima During
the Floral Transition Stage Induced by
Short-Day Photoperiod.
Front. Plant Sci. 12:715683.
doi: 10.3389/fpls.2021.715683
Morphological, Physiological, and
Molecular Responses of Sweetly
Fragrant Luculia gratissima During
the Floral Transition Stage Induced
by Short-Day Photoperiod
XiongfangLiu1,2, YoumingWan1
*, JingAn1, XiujiaoZhang1, YurongCao1, ZhenghongLi1
*,
XiuxianLiu1 and HongMa1
*
1 Research Institute of Resources Insects, Chinese Academy of Forestry, Kunming, China, 2 College of Forestry,
Nanjing Forestry University, Nanjing, China
Photoperiod-regulated oral transition is vital to the owering plant. Luculia gratissima
“Xiangfei” is a owering ornamental plant with high development potential economically and
is a short-day woody perennial. However, the genetic regulation of short-day-induced oral
transition in L. gratissima is unclear. To systematically research the responses of L. gratissima
during this process, dynamic changes in morphology, physiology, and transcript levels were
observed and identied in different developmental stages of long-day- and short-day-treated
L. gratissima plants. Wefound that oral transition in L. gratissima occurred 10d after
short-day induction, but ower bud differentiation did not occur at any stage under long-day
conditions. A total of 1,226 differentially expressed genes were identied, of which 146
genes were associated with owering pathways of sugar, phytohormones, photoperiod,
ambient temperature, and aging signals, as well as oral integrator and meristem identity
genes. The trehalose-6-phosphate signal positively modulated oral transition by interacting
with SQUAMOSA PROMOTER-BINDING-LIKE PROTEIN 4 (SPL4) in the aging pathway.
Endogenous gibberellin, abscisic acid, cytokinin, and jasmonic acid promoted oral transition,
whereas strigolactone inhibited it. In the photoperiod pathway, FD, CONSTANS-LIKE 12,
and nuclear factors Y positively controlled oral transition, whereas PSEUDO-RESPONSE
REGULATOR 7, FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1, and LUX negatively
regulated it. SPL4 and pEARLI1 positively affected floral transition. Suppressor of
Overexpression of Constans 1 and AGAMOUSLIKE24 integrated multiple owering signals
to modulate the expression of FRUITFULL/AGL8, AP1, LEAFY, SEPALLATAs, SHORT
VEGETATIVE PHASE, and TERMINAL FLOWER 1, thereby regulating oral transition. Finally,
wepropose a regulatory network model for short-day-induced oral transition in L. gratissima.
This study improves our understanding of owering time regulation in L. gratissima and
provides knowledge for its production and commercialization.
Keywords: Luculia gratissima, oral transition, photoperiod, owering pathway, phytohormone, regulatory
network
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 2 August 2021 | Volume 12 | Article 715683
INTRODUCTION
Floral transition (the switch from vegetative to reproductive
development) is a critical stage in the life history of owering
plants, particularly in horticultural ornamental plants (Cho
et al., 2017; Shang et al., 2020). is process is regulated by
both environmental and endogenous signals (Cho etal., 2017).
Recently, major breakthroughs have been made in research
on the molecular regulatory networks of oral transition in
Arabidopsis thaliana (Cruciferae), an annual long-day (LD)
photoperiod responsive plant (Liu et al., 2020; Zhang et al.,
2020; Lv et al., 2021). In A. thaliana, dierent endogenous
(autonomous, gibberellin, circadian rhythm, age, and sugar
signals) and environmental (vernalization, temperature, and
photoperiod) signals congregate on some oral integrators,
such as SUPPRESSOR OF OVEREXPRESSION OF CONSTANS
1 (SOC1), FLOWERING LOCUS T (FT), and AGAMOUSLIKE24
(AGL24), further activating oral meristem identity genes,
such as LEAFY (LFY) and APETALA1 (AP1), which irreversibly
convert vegetative meristem to oral meristem (Blümel etal.,
2015). However, there is still much to learn regarding the
regulation of oral transition in perennial woody plants.
Perennial woody plants do not die aer owering. Instead,
they produce new ower buds and vegetative branches annually
and have characteristics of long reproductive cycles and
seasonal owering (Khan et al., 2014). erefore, studies on
annual plants cannot completely reveal the oral transition
mechanisms in perennial woody plants. ere are signicant
dierences in the molecular mechanisms of oral transition
in perennial woody plants compared with those of A. thaliana.
For example, gibberellin (GA) promotes the transition from
vegetative to reproductive development in A. thaliana but
has inhibitory eects in some perennial woody plants
(Yamaguchi et al., 2014; Li et al., 2018; Bao et al., 2020).
Furthermore, in the study on oral transition mechanisms
regulated by light intensity, in contrast to Arabidopsis, which
is aected by retrograde signaling from in response to
photosynthesis (Feng etal., 2016), cultivated roses are specically
controlled by some light-sensitive transcription factor complexes
(Balcerowicz, 2021; Sun et al., 2021). erefore, it is crucial
to accelerate the pace of research on oral transition in
perennial woody plants, which is expected to improve our
understanding of the dierences in oral transition mechanisms
in owering plants with dierent life histories.
Luculia gratissima (Wall.) Sweet (Rubiaceae) is a perennial
evergreen shrub or small tree that is distributed in the
southeastern edge of the Tibetan plateau in southwest China
and neighboring Nepal and Myanmar (Zhou et al., 2011).
L. gratissima “Xiangfei,” a new cultivar cultivated by our research
team for many years, has pink owers, a strong fragrance,
and a large and dense inorescence (Figures 1A,B); it is a
woody horticultural ower with great ornamental value and
economic development potential. In natural conditions, seed-
derived plants of the cultivar “Xiangfei” grow for 2years before
owering, with owering from August to December every year.
However, this plant has not entered the large-scale commercial
production stage because of imperfect owering time regulation
techniques. Previous studies showed that the cultivar “Xiangfei”
can only complete oral transition at short-day (SD) photoperiods
(Wan et al., 2018), and thus, controlling day length to induce
owering is required to achieve year-round production. e
species of interest, L. gratissima, is in a dierent clade than
that of A. thaliana. us, mechanistic dierences are likely to
exist. erefore, understanding the mechanisms of short-day-
induced oral transition in L. gratissima “Xiangfei” has important
signicance for understanding and solving owering-
related problems.
In the present study, we investigated responses of
L. gratissima during short-day-induced oral transition stage
at the morphological, physiological, and transcriptome levels.
e aims of this study were as follows: (1) to observe shoot
apexes of L. gratissima of short-day treatment during ve
developmental stages using morphological and histological
methods to identify the time point of oral transition in
L. gratissima; (2) to measure endogenous substance contents
to study the soluble sugar and hormone eects in oral
transition in L. gratissima; and (3) to conduct an RNA
sequencing (RNA-seq) analysis of the transcriptomes of
L. gratissima shoot apexes and leaves at four dierent stages,
7, 10, 13, and 19 days aer the initiation of long-day (LD)
and short-day (SD) treatments, to study the molecular regulatory
mechanism of short-day-induced oral transition in
L. gratissima. e results presented in this research will aid
in regulating L. gratissima owering and achieving year-round
production. Additionally, identication of important regulatory
genes will provide important guidance for owering-related
molecular breeding in the future.
MATERIALS AND METHODS
Plant Materials, Growth Conditions, and
Light Treatments
Luculia gratissima cultivar “Xiangfei” cuttings from three-
year-old plants were obtained from the central Yunnan Plateau
experimental station of Research Institute of Resources Insects,
Chinese Academy of Forestry (Yunnan, China; 25°13'N,
102°12'E, 1826 m a.s.l.). In mid-December 2016, cuttings
with two stem nodes and shoot apexes were planted in a
mixed matrix (peat and perlite at a 3:1 ratio) and grown
in an 18–25°C greenhouse under natural lighting. Cuttings
with roots were transplanted into pots and maintained in
the same greenhouse under natural lighting. To prevent these
plants from being induced by SD photoperiod, shoot apical
meristems (SAMs) were removed from all plants when 2–3
new stem nodes were formed, and high-pressure sodium
lamps were used for additional lighting during 22:00–02:00
(night-break treatment; Figure1C). In addition, considering
the eects of individual developmental age on owering time
(Evans et al., 1992), some plants were placed in the natural
environment as controls and the time when ower bud
dierentiation occurred in these plants was used as the start
time for photoperiod treatments. On 10 August 2017 (when
ower buds began to appear in some natural control plants),
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 3 August 2021 | Volume 12 | Article 715683
plants with the same number of branches longer than 5 cm
were selected from among the night-break treatment plants
and then were subjected to either LD (night-break treatment
as described above) or SD (10h light/14h dark; Figure1D)
for a further 90 days. e light source was supplied using
high-pressure sodium lamps. e greenhouse temperature
was 20 ± 2°C with approximately 60% relative humidity.
Shoot apexes and their surrounding leaves of the main
branches of SD and LD plants were sampled during 09:00–
11:30 every 3–5 d aer the initiation of the photoperiod
treatments. For each stage, 10–20 shoot apexes and their
surrounding leaves were packed together into each of the
10 biological replicates, of which one biological replicate
was rapidly immersed into FAA xative (50% ethanol: acetic
acid: formaldehyde, 18:1:1) for morphological analysis, whereas
the remaining nine biological replicates were snap-frozen in
liquid nitrogen and then stored at −80°C for measurements
of soluble sugar and endogenous hormone contents, as well
as RNA extraction.
Morphological Anatomical Observations
Ten FAA-xed shoot apexes of SD and LD plants at each
stage were made into sections with a thickness of 8–10μm
using paran section method (Fischer et al., 2008), and
were stained with safranin O-fast green, and then were
mounted with neutral resin. Finally, the process of bud
development was observed under a Carl Zeiss Axio Scope
A1 Microscope (Carl Zeiss Microscopy GmbH, Göttingen,
Germany).
Measurements of Soluble Sugar and
Endogenous Hormone Contents
According to the anatomical observation results, samples
from the SD treatment at five stages [0d (SD0), 7 d (SD7),
10 d (SD10), 13 d (SD13), and 19 d (SD19)] close to
flower bud differentiation (Figure 2) were selected for
measurements of soluble sugar and endogenous hormone
contents of three biological replicates. For each of the three
biological replicates from each stage, soluble sugar contents
were measured using sulfuric acid-anthrone colorimetric
assays as previously reported (Wang et al., 2015), and
endogenous hormones [GA3, indoleacetic acid (IAA), ABA,
and zeatin (ZT)] were quantified with high-performance
liquid chromatography-mass spectrometry (Aglient1290,
Nanjing, China; AB 6500, Nanjing, China) as previously
FIGURE1 | Features of Luculia gratissima “Xiangfei” and the overview of greenhouses under two different photoperiods. (A) Whole plant of L. gratissima “Xiangfei.”
(B) Flowers of L. gratissima “Xiangfei.” (C) Greenhouse under night-break treatment. (D) Greenhouse under short-day photoperiod.
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 4 August 2021 | Volume 12 | Article 715683
reported (Pan et al., 2010). Before comparing changes in
the soluble sugar and hormone contents among the five
stages, the Shapiro–Wilk test and Levene test were used
to analyze the normality and homogeneity of variance of
each dataset. Because the four sets of data did not follow
a normal distribution (p < 0.05), a Kruskal-Wallis H test
was employed for analysis of significant differences, and
false discovery rate (Benjamini and Yekutieli, 2001) was
used for the multiple testing correction of significant p-values.
Additionally, the Tukey–Kramer method was used for post-hoc
testing of soluble sugar and hormone contents at the five
stages. The above analyses were performed in the “car”
and “stats” packages in R software and the data were
expressed as the mean ± SD.
Transcriptome Sequencing and Data
Analysis
Likewise, based on the anatomical observation results, samples
from the SD and LD treatments at the four stages [7 d (SD7
or LD7), 10 d (SD10 or LD10), 13 d (SD13 or LD13), and
19 d (SD19 or LD19)] close to ower bud dierentiation of
SD plants (Figure 2) were selected for RNA extraction. Total
RNA extracted from each of the three biological replicates
was divided into two parts, of which one was used for RNA-seq
and the other was used for quantitative real-time PCR (qRT-
PCR) validation. Total RNA was extracted with the plant total
RNA Kit (Tiangen, Beijing, China) following manufacturer’s
instructions. e cDNA library construction and paired-end
sequencing were conducted with an Illumina HiSeq™ 4,000
AB C
DE F
GH I
JKL
FIGURE2 | Luculia gratissima morphological and histological characteristics, shoot apexes at ve time points upon short-day treatment. (A–C) Vegetative buds in
the undifferentiated stage (SD0 to SD7). (D–F) Bract primordial differentiation stage (SD10). (G–I) Inorescence primordial differentiation stage (SD13). (J–L) Floret
primordial differentiation stage (SD19). (A,B,D,G,H) Histological images obtained from parafn-embedded sectioned samples (scale bar: 100μm). (E,J,K)
Histological images obtained from parafn-embedded sectioned samples (scale bar: 50μm). (C,F,I,L) The external morphology of shoot apexes at different
developmental stages (scale bar: 5mm). BP, bract primordia; FP, oret primordia; IP, inorescence primordia; LIP, lateral inorescence primordium; LP, leaf primordia;
and VC, vegetative cone.
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 5 August 2021 | Volume 12 | Article 715683
(Illumina, San Diego, California, United States) at the Gene
Denovo Biotechnology Company (Guangzhou, China). e
generated raw reads were ltered by removing adapter sequences
and ambiguous reads (N> 10%) and low-quality reads (more
than 40% of bases with value of Q≤ 20) to obtain high-
quality clean reads. Without reference genome, clean reads
were de novo assembled as a transcriptome reference database
for L. gratissima via Trinity soware (Grabherr et al., 2011).
Furthermore, clean reads were mapped to ribosome RNA
(rRNA) to identify residual rRNA reads. e rRNA removed
reads were further mapped to the reference transcriptome using
short reads alignment tool Bowtie2 (Langmead et al., 2009)
by default parameters. e reference transcriptome unigenes
without rRNA reads were generated for next analysis.
All non-redundant unigenes were aligned with selected
cutos of value of E≤1e-05 to six protein databases, including
the NR (the NCBI non-redundant protein databases), KOG
(EuKaryotic Orthologous Groups), Kyoto Encyclopedia of Genes
and Genomes, Swiss-Prot, evolutionary genealogy of genes:
Non-supervised Orthologous Groups, and Protein families
database of alignments and hidden Markov models. Based on
the NR annotation results, these unigenes were also annotated
for GO (Gene Ontology) using the Blast2GO soware (Conesa
etal., 2005), and then GO functional classication of unigenes
was obtained by the WEGO soware (Ye et al., 2006).
qRT-PCR Analysis
qRT-PCR was conducted on nine owering-related unigenes
in this study, including COP1 (Unigene0031506), ZTL
(Unigene0041339), FKF1 (Unigene0038380), GI (Unigene0051409),
ELF3 (Unigene0051761), PRR1 (Unigene0045946), PRR7
(Unigene0003564), PRR5 (Unigene0047475), and LHY
(Unigene0035686). To accurately measure gene expression levels,
the ACT7/EF1-α combination obtained from the past screening
was used as an internal reference gene for standardization and
correction (Supplementary Data). Primer3 soware (Rozen and
Skaletsky, 2000) was used to design specic primers for each
gene (Supplementary Table S1). e KR106 FastQuantity RT
Kit (with gDNase; Tiangen, Beijing, China) was used for reverse
transcription of 1 μg total RNA into cDNA according to the
manufacturer’s instructions. e StepOnePlus™ Real-Time PCR
System (ermo Scientic, Wilmington, DE, United States) was
used for qRT-PCR in a 20 μl reaction system, including 4 μl
of 50ng cDNA template, 10μl of 2×qPCR Master Mix (Tiangen,
Beijing, China), 0.4μl each of 10μm forward and reverse primers,
and 5.2 μl ddH2O. e qRT-PCR amplication conditions were
as follows: pre-denaturation at 95°C for 90s, followed by 40cycles
of denaturation at 95°C for 5 s, annealing at 60°C for 15 s,
and extension at 72°C for 20 s, followed by a nal extension
step at 72°C for 5 min, aer amplication, a 65–95°C melting
curve analysis was conducted to measure product specicity. e
2−ΔΔC method (Livak and Schmittgen, 2001) was used to calculate
the relative expression levels of the genes in the qRT-PCR
experiment. e normalization of gene expression was conducted
using the geometric mean of two internal reference genes, ACT7
and EF1-α (Vandesompele et al., 2002).
Identication and Functional Enrichment
of DEGs
e Reads Per kb per Million reads (RPKM) method was
used to evaluate unigene expression levels (Mortazavi et al.,
2008). Pairwise comparisons were conducted between LD and
SD samples to identify dierentially expressed genes (DEGs)
in response to SD photoperiod during the oral transition
process in L. gratissima. To generate accurate log2foldchange
estimates, EdgeR package version 3.8 (Robinson et al., 2010)
was used. e thresholds for dierential expression were set
at fold change 2 (log2foldchange=1) and FDR value cuto 0.05.
e Mercator online tool1 was employed for gene function
predictions for the DEGs with a BLAST-CUTOFF of 50. e
obtained mapping les were uploaded to MapMan version 3.6
(imm et al., 2004) for the functional analysis of DEGs.
Wilcoxon rank-sum test was used to analyze the log2foldchange
of DEGs in each comparison before MapMan version 3.6
(imm et al., 2004) was used for visualization of the results.
Co-expression Network Analysis
Weighted gene co-expression network analysis (WGCNA;
Langfelder and Horvath, 2008) was employed to generate the
co-expression network modules of DEGs. e parameter settings
used were so threshold = 20, minModuleSize = 30,
TOMType = signed, and mergeCutHeight = 0.25, and default
values were used for the remaining parameters. e eigengene
value of every module was calculated and the associations between
every gene in eight samples were tested. KOBAS 3.0 (Xie et al.,
2011) was used for GO enrichment analysis of genes in the
clustering modules. Cytoscape version 3.7.1 (Shannon et al.,
2003) was used for visualization of the co-expression network.
RESULTS
Morphological Differentiation of Shoot
Apexes During Floral Transition
Luculia gratissima cultivar “Xiangfei” cuttings from three-year-
old plants were planted and grown for about 8months before
photoperiod treatments. When some ower buds appeared in
natural control plants, the generated cutting plants were
transferred to SD conditions (10 h light/14 h dark, 20 ± 2°C,
60% relative humidity) or LD conditions (night-break treatment
for 4 h, 20 ± 2°C, 60% relative humidity). Shoot apexes and
their surrounding leaves of the main branches of SD and LD
plants were sampled during 09:00–11:30 every 3–5 d aer the
initiation of the photoperiod treatments.
e morphological dierentiation of L. gratissima shoot
apexes was observed through paran sections. e results
showed that 0d to 7 d under the SD treatment (SD0 to SD7)
was the vegetative growth stage (undierentiated stage), in
which the tip of the growth cone in the bud was narrow and
pointed and surrounded by leaf primordia (Figures 2A–C).
At 10 d aer the initiation of the SD treatment (SD10), the
1
https://www.plabipd.de/portal/mercator-sequence-annotation
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 6 August 2021 | Volume 12 | Article 715683
bract primordial dierentiation stage began (Figures 2D–F).
In this stage, the growth cone of the bud appeared dome
shape; subsequently, the dome-shaped growth cone began
broadening and attening, and the bract primordia along the
periphery were formed, which was an important marker of
the transition from vegetative growth to reproductive growth
(Figures2D–F). At 13d aer the initiation of the SD treatment
(SD13), the inorescence primordial dierentiation stage began.
At this stage, the growth cone in the bract primordia elongated
to form three hemispherical protrusions, i.e., inorescence
primordia. Simultaneously, the lateral base of the bract primordia
dierentiated into lateral inorescence primordia. Next, bilateral
protrusions at each hemispherical inorescence primordium
dierentiated into bract inorescences (Figures2G–I). At 19d
aer the initiation of the SD treatment (SD19), the oret
primordial dierentiation stage began and a single inorescence
primordium in the bract primordia gradually widened to become
oret primordia at the tip of the bud (Figures 2J–L). ese
results showed that the oral transition period began 10 d
aer the initiation of the SD treatment, and the selection of
time points before and aer this period could facilitate the
physiological study of oral transition. However, the buds of
LD plants were at vegetative growth stage all the time
(Supplementary Figure S1). erefore, the LD treatment was
used as a control in this study, and 7 d (SD7), 10 d (SD10),
13 d (SD13), and 19 d (SD19) in the SD treatment were
selected to study the physiological and molecular regulation
patterns of oral transition. LD samples (i.e., LD7, LD10, LD13,
and LD19) for RNA-seq analysis were taken in parallel at the
same time points as respective SD samples.
Dynamic Changes in Endogenous
Substance Content During Floral
Transition
Contents of soluble sugars and endogenous hormones [gibberellin
(GA3), IAA, abscisic acid (ABA), and zeatin (ZT)] were measured
at 0 d (SD0), 7 d (SD7), 10 d (SD10), 13 d (SD13), and 19 d
(SD19) aer the initiation of the SD treatment. e Kruskal-
Wallis H test results showed that except for GA3, which could
not be detected because it was below the limit of quantitation
(0.1 ng/ml), there were signicant dierences in the contents
of the other substances among the ve stages (adjusted p<0.05;
Figure 3). Soluble sugar, ZT, and IAA reached their peaks at
SD0, which were 28.86 ± 0.67 mg g−1 FW, 2.15 ± 0.30 ng g−1
FW, and 0.69 ± 0.04 ng g−1 FW, respectively. Additionally,
soluble sugar and ZT decreased from SD0 to SD19, indicating
that the soluble sugar and ZT contents in SAMs of L. gratissima
were maintained at a relatively low level during the owering
process. Interestingly, IAA showed an increase in SD13 before
decreasing. Similarly, ABA initially increased from SD0 to SD13
and subsequently declined.
Additionally, post-hoc results showed that there were
extremely signicant dierences in the pairwise comparisons
between the ve time points for ABA (p <0.001). IAA only
showed no signicant dierences between SD7 and SD13
(p>0.1). Soluble sugar did not show any signicant dierences
between SD0 and SD7 (p > 0.1). ZT did not show any
signicant dierences between SD0 and SD10, between SD7
and SD10, or between SD13 and SD19 (p> 0.1). From these
results, it can be seen that ABA levels changed rapidly and
dynamically over these ve stages, whereas ZT levels exhibited
little change over the same period. Changes in soluble sugar
content mainly occurred in later periods (SD13 to SD19).
In contrast to these substances, IAA changes were relatively
constant (Figure 3).
RNA-Seq and qRT-PCR Identication of
DEGs
Transcriptomes were generated for three biological replicates
from the SD and LD treatments at each of the time points
corresponding to the four stages of bud dierentiation in
SD treatment plants, i.e., at 7 d, 10 d, 13 d, and 19 d
(Figure 2), yielding a total of 24 transcriptomes. A total of
1,236,426,670 raw sequencing reads were generated from 24
samples, 1.2 × 109 high-quality clean reads (181Gb) were
obtained aer ltering, with mean Q20, Q30, and GC contents
of 99.11, 97.18, and 43.53%, respectively
(Supplementary Table S2). A total of 79,870 unigenes (≥
200 b) were generated from de novo assembly, and the N50
length was 2,118bp (Supplementary Table S3). Among these
unigenes, 35,725 unigenes (44.73%) were successfully annotated
to at least one database (Supplementary Figure S2).
With RNA from the same 24 samples used for transcriptome
generation, qRT-PCR was conducted for nine owering-
related unigenes identied in through RNA-seq, including
COP1 (Unigene0031506), ZTL (Unigene0041339),
FKF1 (Unigene0038380), GI (Unigene0051409), ELF3
(Unigene0051761), PRR1 (Unigene0045946), PRR7 (Unigene
0003564), PRR5 (Unigene0047475), and LHY (Unigene0035686).
e results of qRT-PCR showed that except for PRR5
(Unigene0047475), the expression patterns of the other eight
genes were generally consistent with the RNA-seq data
(Supplementary Figure S3), indicating that the transcriptome
data generated in this study were reliable and valid. e
inconsistency between the relative expression and RPKM
values of PRR5 occurred in LD7 and SD7 samples, and for
the possible reasons of this inconsistency, on the one hand,
it could be that PRR5 was not a DEG in the RNA-seq data,
and on the other hand, the RPKM values of PRR5 were
lower than 10in both LD7 and SD7 samples, in which there
could be false positives.
A total of 113 (SD7-vs.-LD7), 420 (SD10-vs.-LD10), 483
(SD13-vs.-LD13), and 464 (SD19-vs.-LD19) DEGs were obtained
by comparing the LD and SD treatments
(Supplementary Figure S4 and Table S4). A total of 1,226
DEGs were identied from these four comparisons, of which
ve DEGs were shared by four comparisons, and 250 DEGs
were present in more than one comparison. ere were 110,
302, 288, and 276 stage-specic DEGs in SD7-vs.-LD7, SD10-
vs.-LD10, SD13-vs.-LD13, and SD19-vs.-LD19, respectively
(Supplementary Figure S4).
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 7 August 2021 | Volume 12 | Article 715683
Functional Classications of DEGs
MapMan is an eective tool for systematic analysis of plant
transcriptome metabolic pathways and other biological processes
(Ramšak et al., 2013). We employed MapMan to overview
transcriptional changes in regulatory, metabolic, and cellular
response-related genes (Supplementary Table S5). In “regulation
overview,” more DEGs were detected in the other three comparisons
contrasted with SD7-vs.-LD7, showing that the physiological and
molecular characteristics aer ower bud dierentiation (SD10,
SD13, and SD19) were signicantly dierent from that before
ower bud dierentiation (SD7). In the IAA metabolic subclass,
more DEGs were upregulated in SD19-vs.-LD19 compared with
SD7-vs.-LD7, SD10-vs.-LD10, and SD13-vs.-LD13
(Supplementary Figure S5). Yet, IAA content increased from
SD10 to SD13 to continue decreasing aerward. Anyhow, the
dierences between dates were small, although signicant (Figure3).
erefore, IAA was not a key factor mediating oral transition
in L. gratissima. ABA metabolism-related DEGs were signicantly
upregulated in all four comparisons (Supplementary Figure S5),
and ABA levels were overall increasing in the process of oral
transition (Figure 3), demonstrating that ABA could promote
oral transition in L. gratissima. In “minor CHO metabolism”,
trehalose biosynthesis-related DEGs were only upregulated in
SD7-vs.-LD7 (Supplementary Figure S6). “Cellular response
overview” showed that more development-related DEGs were
upregulated in SD10-vs.-LD10 compared with the other three
comparisons (Supplementary Figure S7), indicating that these
DEGs promoted oral transition in L. gratissima.
Co-expression Module Analysis for DEGs
WGCNA is a systems biology method for analyzing the correlation
relationships between genes in multiple samples (Langfelder
FIGURE3 | Luculia gratissima endogenous soluble sugar content and hormonal changes, shoot apexes and leaves at ve stages upon short-day treatment. The
y-axis shows soluble sugar and four hormones, and the x-axis shows the average relative abundance of the endogenous soluble sugars and hormones. Colored
columns represent different developmental stages. *0.01<p≤0.05; **0.001<p≤0.01.
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 8 August 2021 | Volume 12 | Article 715683
and Horvath, 2008). In this study, the results of WGCNA
showed that 1,226 DEGs in eight samples were clustered in
11 dierent co-expression modules (labeled with dierent colors;
Figure 4A). It is noteworthy that four out of 11 co-expression
modules signicantly correlated with a single sample (r >0.9,
p<0.05; Figure4B and Supplementary Table S6). For example,
the largest module (black module) included 247 (20.15%)
SD19-specic DEGs (Figure4B and Supplementary Table S6A).
We further conducted GO enrichment analysis on 11
co-expression modules, and only the greenyellow module was
not signicantly enriched for any GO terms
(Supplementary Table S7). Some GO terms were specically
identied in only a single module. For example, 120 specic
GO terms were identied in the black module, which mainly
involved signal transduction and negative regulation of metabolic
processes, and 34 module-specic GO terms were identied
in the brown module, which was mainly associated with growth
and development (Supplementary Table S7). However, several
GO terms, including “response to organic substance” and
“response to a stimulus,” appeared in multiple modules
(Supplementary Table S7), indicating possible module-gene
interactions. Overall, the extensively enriched GO terms showed
that multiple biological processes were involved in the oral
transition in L. gratissima.
e 11 modules were divided into seven categories based
on the correlations between modules (Figure 4C). e heat
map showed that there was a high correlation between the
blue, magenta, pink, and tan modules, in which the genes
were highly expressed in SD7 and SD10 (Figures 4B,C), and
were signicantly enriched in multiple GO terms involving
secondary metabolite biosynthesis, signal transduction, and
regulation of developmental processes (Supplementary Table S7).
Identication of DEG Expression Patterns
Associated With Floral Transition in
L.gratissima
According to the above functional classications and WGCNA
of these DEGs, and owering-related genes previously reported
in model plants (such as A. thaliana; Blümel et al., 2015; Bao
etal., 2020), a total of 146 unigenes were identied as homologous
genes related to oral transition in L. gratissima, involving
several owering pathways: sugar metabolism, hormone
metabolism and signal transduction, photoperiod, ambient
temperature, aging pathways, oral integrator, and oral meristem
identity genes. Among these oral transition-related homologous
genes, stage-specic DEGs, and common DEGs in SD7-vs.-LD7,
SD10-vs.-LD10, SD13-vs.-LD13, and SD19-vs.-LD19, are listed
in Supplementary Table S8.
The Expression Pattern of Sugar Signal-
Related Homologs
e sugar signal pathway, which responds to the sugar budget
in plants, is one of the important pathways mediating the
transition from vegetative to oral meristems (Blümel etal.,
2015). A total of 29 (19.86%) DEGs associated with sugar
signal-related genes were identied, involving 23 sugar signal-
related homologs. ese genes expressed dierently in dierent
development stages of L. gratissima. For example, HEXOKINASE
(HK) homologs (Unigene0044869 and Unigene0044870) were
signicantly upregulated in SD7-vs.-LD7 and SD13-vs.-LD13,
and a BETA-GLUCOSIDASE 24 homolog (Unigene0013088)
was signicantly upregulated in SD10-vs.-LD10. Meanwhile,
Unigene0009721 and Unigene0041893, homologs of
GALACTINOL SYNTHASE 2 and RAFFINOSE SYNTHASE
participating in ranose synthesis, were upregulated in
SD7-vs.-LD7. In addition, TREHALOSE-6-PHOSPHATE
SYNTHSE (TPS) homologs (Unigene0019787, Unigene0024389,
Unigene0013555, Unigene0054604, Unigene0004913, and
Unigene0062998) were upregulated at various stages, and
SWEET16 homolog (Unigene0012661) was signicantly
upregulated in SD7-vs.-LD7 and SD10-vs.-LD10 (Figure 5E
and Supplementary Table S9). Hence, these genes may
directly or indirectly participate in oral transition in L.
gratissima.
The Expression Patterns of Phytohormone
Metabolism and Signal Transduction
Homologs
Many studies have demonstrated that various phytohormones
participate in the regulation of oral transition (Shu etal., 2018;
Lin et al., 2019; Zhang et al., 2019; Bao et al., 2020). A total
of 20 (13.70%) DEGs associated with phytohormone metabolism
were identied, and these involved 16 phytohormone metabolism
homologous genes and were related to nine phytohormone
metabolism pathways. Among these genes, GIBBERELLIN 2-BETA-
DIOXYGENASE 1 (GA2OX1) homologs (Unigene0030732) and
GA2OX8 homologs (Unigene0073113), which are involved in
GA metabolism, were signicantly upregulated in SD10-vs.-LD10
and/or SD19-vs.-LD19. Meanwhile, Unigene0034382 (CYP707A1
homolog) and Unigene0042754 and Unigene0042755 (NCED1
homologs), respectively, encoding abscisic acid (ABA)
8'-hydroxylase 1 and nine-cis-epoxycarotenoid dioxygenase, were
signicantly upregulated in SD10-vs.-LD10. In addition, a homolog
(Unigene0035296) of YUC4, encoding indole-3-pyruvate
monooxygenase, which mediates auxin biosynthesis, was
signicantly upregulated in SD19-vs.-LD19. Additionally, genes
encoding cytokinin (CK) dehydrogenase 7 (CKX7;
Unigene0036599) and cytokinin dehydrogenase (CYP735A1;
Unigene0029738) were signicantly downregulated in SD19-
vs.-LD19. CYTOCHROME P450 734A1 homolog
(Unigene0036368), which participates in brassinolide (BR)
biosynthesis, was upregulated in SD10-vs.-LD10 and SD19-
vs.-LD19; the jasmonate (JA) metabolism-related JA S M O NATE
O-METHYLTRANSFERASE homolog (Unigene0020912) and the
salicylic acid (SA) metabolism-related UDP-GLYCOSYLTRAN
SFERASE 74F1 homolog (Unigene0004033) were downregulated
in SD19-vs.-LD19. A homolog of CAROTENOID CLEAVAGE
DIOXYGENASE 7 (CCD7, Unigene0069349) involving in
strigolactone (SL) biosynthesis was also identied and showed
signicant downregulation in SD10-vs.-LD10 (Figure 5C and
Supplementary Table S9).
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 9 August 2021 | Volume 12 | Article 715683
A
BC
FIGURE4 | Weighted co-expression network analysis of 1,226 DEGs at four developmental stages of L. gratissima, short- or long-day treatments. (A) Hierarchical
cluster tree showing the co-expression modules, with each tree leaf representing one gene. The major tree branches constitute 11 modules labeled by different
colors. (B) Heat map of gene relative expression of different modules (y-axis) in eight samples (x-axis). The Z-score normalized RPKM value for an individual gene at
a given developmental stage is indicated in a green (low expression) to red (high expression) scale. (C) Eigengene network representing the relationships among the
different modules. The hierarchical clustering dendrogram of the eigengenes shows the relationships among the modules, whereas the heat map shows the
correlation between the different modules, with deeper red color representing a stronger correlation.
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 10 August 2021 | Volume 12 | Article 715683
A total of 39 (25.85%) DEGs associated with phytohormone
signal transduction of nine hormones were identied and
involved 30 phytohormone signal transduction homologs that
were associated with signal transduction for nine hormones.
Among these DEGs, GID1B homologs (Unigene0032780,
Unigene0032781, and Unigene0063035), encoding a gibberellin
receptor, were upregulated in SD10-vs.-LD10, whereas an RGL3
homolog (Unigene0071862), encoding a DELLA protein, was
signicantly downregulated in SD19-vs.-LD19. e ABA signal
transduction-related EID1-LIKE F-BOX PROTEIN 3 (EDL3)
homolog (Unigene0018152) was upregulated in SD10-vs.-LD10,
and SAUR71 homologs (Unigene0021953 and Unigene0025106),
encoding the auxin-responsive protein, were upregulated in
SD13-vs.-LD13 and SD19-vs.-LD19. Moreover, in the CK
signaling pathway, homologs of AHPs (Unigene0034629,
Unigene0004315, and Unigene0034630), encoding histidine-
containing phosphotransfer protein, were highly expressed in
SD10, SD13, and SD19, and an ARR6 homolog (Unigene0049441),
encoding a two-component response regulator, was upregulated
in SD19-vs.-LD19. In addition, a BRI1 homolog
(Unigene0024976) in the BR signaling pathway was signicantly
upregulated in SD7-vs.-LD7; homologs of MYC4
(Unigene0009399) and TIFYs (Unigene0022959 and
Unigene0019294) in the JA signaling pathway were upregulated
in SD10-vs.-LD10; and a DWARF14 (D14) homolog
(Unigene0028658), participating in SL signal transduction, was
upregulated in SD7-vs.-LD7 but downregulated in SD19-vs.-LD19
(Figure 5D and Supplementary Table S9).
Expression Patterns of Genes Associated
With Photoperiod Pathways
e photoperiod owering pathways in plants include the
photosensory pathway, the circadian clock, and the systemic
eector (Nelson etal., 2009). A total of 10 (6.84%) photoperiod-
related homologs were identied. Among these homologs,
A
B
CE
F
G
H
D
FIGURE5 | Expression proles of genes associated with L. gratissima oral transition at four developmental stages, short- or long-day treatments. Relative
expression prole of (A) photoperiod pathway-related genes, (B) ambient temperature pathway-related genes, (C) phytohormone metabolism-related genes,
(D) phytohormone signal transduction-related genes, (E) sugar signal-related genes, (F) aging pathway-related genes, (G) oral integrator-related genes, and
(H) oral meristem identity genes. The Z-score normalized RPKM value for an individual gene at a given developmental stage is represented in a green (low
expression) to red (high expression) scale.
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 11 August 2021 | Volume 12 | Article 715683
CHLOROPHYLL A-B BINDING PROTEIN (Unigene0075619)
was downregulated in SD19-vs.-LD19, whereas CONSTANS-
LIKE 12 (COL12, Unigene0039617) and FD (Unigene0027311)
were upregulated in SD13-vs.-LD13. Meanwhile, homologs of
the FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1
(FKF1, Unigene0038380) and the PRR7 (Unigene0003564) were
both downregulated in SD13-vs.-LD13, and a LUX homolog
(Unigene0011585) was downregulated in SD10-vs.-LD10, whereas
homologs of the nuclear factor Y (NF-Ys; Unigene0025001,
Unigene0002375, and Unigene0033157) were upregulated at
one or more stages (Figure 5A and Supplementary Table S9).
Expression Patterns of Genes Associated
With the Ambient Temperature Pathway
Plant responses to photoperiod and temperature are coupled
(Dong etal., 2020; Meng et al., 2020). e photoperiod-induced
oral transition could also aect the expression of a series of
ambient temperature-related genes in plants. We identied 28
(19.18%) ambient temperature-related DEGs involving 18 homologs,
primarily including the HEAT STRESS TRANSCRIPTION
FACTORS, HEAT SHOCK PROTEIN/COGNATE (HSPs), and
pEARLI1, most of which were highly expressed at several stages
under LD (Figure 5B and Supplementary Table S9).
Expression Patterns of Aging Pathway-
Related, Floral Integrator, and Floral
Meristem Identity Genes
e aging pathway is an endogenous owering pathway in
plants (Yao et al., 2019). SQUAMOSA PROMOTER-BINDING-
LIKE PROTEIN 4 (SPL4) homologs (Unigene0024429 and
Unigene0024430) in the aging pathway were upregulated in
SD10-vs.-LD10, SD13-vs.-LD13, and SD19-vs.-LD19 (Figure5F
and Supplementary Table S9).
Floral integrators combine environmental and endogenous
signals to mediate owering in plants (Blümel et al., 2015).
e oral integrator gene SOC1 homologs (Unigene0039572
and Unigene0039575) were upregulated in SD10-vs.-LD10,
SD13-vs.-LD13, and SD19-vs.-LD19, whereas the AGL24 homolog
(Unigene0049016) was downregulated in SD19-vs.-LD19
(Figure 5G and Supplementary Table S9).
Genetic networks regulating oral transition in plants
ultimately activated oral meristem identity genes, thereby causing
the transformation from vegetative to oral meristems
(Gregis et al., 2006). A total of 15 (10.27%) related DEGs were
identied, involving nine oral meristem identity genes
(Supplementary Table S9). Among these genes, homologs of
AGL8/FRUITFULL (FUL; AGL8, also known as FUL;
Unigene0019277, Unigene0004737, Unigene0042052,
Unigene0042053, and Unigene0042058), APETALA 1 (AP1;
Unigene0019278, Unigene0019279, and Unigene0031106), LFY
(Unigene0030979 and Unigene0030980), and SEPALLATAs (SEPs;
Unigene0000607, Unigene0034045, and Unigene0025130) were
upregulated in one or more developmental stages, whereas homologs
of SHORT VEGETATIVE PHASE (SVP, Unigene0049018) and
TERMINAL FLOWER 1 (TFL1, Unigene0026727) were
downregulated in SD19-vs.-LD19, SD10-vs.-LD10, and SD13-
vs.-LD13 (Figure 5H and Supplementary Table S9).
Co-expression Network of Floral
Transition-Related Genes
A co-expression network constructed using 126 oral transition-
related DEGs with edge weights > 0.1 showed 10 hub genes
with great connectivity, including homologs of
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE
(GAPDH, Unigene0005846), AKR1B1 (Unigene0076531), PKM
(Unigene0073914), ENOLASE 1 (Unigene0011083 and
Unigene0011084), MED37E (Unigene0051600), L-LACTATE
DEHYDROGENASE A CHAIN (Unigene0009368), HSP83A
(Unigene0031524), FUL (Unigene0042052), and SEP4
(Unigene0025130; Supplementary Figure S8). e genes with
the highest network degree were GAPDH (Unigene0005846),
AKR1B1 (Unigene0076531), and PKM (Unigene0073914),
which participated in sucrose and starch catabolism
(Supplementary Table S9).
DISCUSSION
e timing of oral transition in plants is jointly regulated
by internal and external environmental cues, of which
photoperiod is one of the major environmental factors that
aect oral transition in plants (Blümel et al., 2015; Chang
et al., 2019). L. gratissima is a horticultural ornamental plant
with high development potential, and therefore, elucidating
the molecular mechanism of its SD photoperiod-induced oral
transition is important to its year-round production for
commercial purposes. In this study, weconducted transcriptome
sequencing of L. gratissima shoot apexes and leaves at four
stages under LD and SD treatments. A total of 79,870 unigenes
were obtained by de novo assembly, of which 49.02% were
successfully annotated. Currently, there is no report on
L. gratissima transcriptome assembly and our assembled and
annotated transcriptome of L. gratissima provides a valuable
genetic resource for breeding this species.
Sugar Signal Mediates Floral Transition in
L. gratissima
Sugars are an important energy source and participate in oral
transition in plants as important signaling molecules (Lebon
et al., 2008; Ortiz-Marchena et al., 2015). In co-expression
network analysis, all of the rst three hub genes (GAPDH,
AKR1B1, and PKM) were related to sugar metabolism, implying
that sugar might play a vital role in the oral transition process
in L. gratissima. Leaves are the primary organ of sugar synthesis
in plants, and SAMs are the sites of sugar mobilization and
consumption, both of which form an important source-sink
unit (Bernier and Périlleux, 2005). Floral transition in plants
is not only directly associated with sugar content from source
and sink but also is regulated by sugar transport (Smeekens
et al., 2010). Previous studies have indicated that source-sink
regulation could be achieved by the interaction between the
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 12 August 2021 | Volume 12 | Article 715683
bidirectional sugar transporter SWEET and the FT-like protein
(Abelenda etal., 2019). In this study, SWEET16 (Unigene0012661)
was signicantly upregulated in SD7-vs.-LD7 and SD10-vs.-LD10
(Figure 5E and Supplementary Table S9), indicating that
SWEET participated in sucrose transport during oral transition
in L. gratissima. However, soluble sugars in SAMs decreased
from SD0 to SD19 (Figure 3), which is not consistent with
the expression prole of genes associated with sucrose
metabolism. We speculated that SAM only synthesized limited
levels of soluble sugar but SWEET16 (Unigene0012661) expression
in SAMs was only high at SD7 and SD10, and its expression
level decreased as SD treatment duration increased (Figure 5E
and Supplementary Table S9), subsequently causing a decrease
in the rate of the sucrose transport from leaves to SAMs; this
suggests that sucrose only acts as an energy source in oral
transition in L. gratissima.
Trehalose-6-phosphate (T6P) is a component of the plant
sugar signaling system and has important eects on owering
and development (Kataya et al., 2020). In A. thaliana, the T6P
pathway in leaves induced the expression of the origen gene
FT in the photoperiodic pathway to aect oral transition,
whereas in SAMs, the expression of SPL in the aging pathway
was controlled by the T6P pathway to directly aect the
expression of oral transition-related genes (Wahl etal., 2013).
erefore, the T6P pathway is an important signal that coordinates
owering induction. In this study, except for the T6P synthase
homolog TPS (Unigene0013555) that was downregulated in
SD19-vs.-LD19, other TPSs were upregulated at one or more
stages during oral transition in L. gratissima (Figure 5E and
Supplementary Table S9), showing that TPS homologs participate
in oral transition in L. gratissima and the T6P signaling
pathway is signicantly enhanced during oral transition. SPL4
was also highly expressed at SD10, demonstrating that T6P
in L. gratissima SAM promoted oral transition by regulating
SPL4 expression. HK acts as a catalytic enzyme to catalyze
hexose phosphorylation, as well as a glucose signal sensor
mediating the interaction between the glucose signaling pathway
and the ABA signaling pathway to regulate plant development
(Moore et al., 2003; Teng et al., 2008). In this study, HK
homologs (Unigene0044869 and Unigene0044870) were
upregulated in SD7-vs.-LD7 and SD13-vs.-LD13 (Figure 5E
and Supplementary Table S9). We speculate that HK mainly
catalyzed hexose phosphorylation to provide an energy source
for initiating oral transition at SD7 and acted as a glucose
signal sensor to participate in L. gratissima ower development
at SD13.
In summary, the sugar metabolism-related genes TPS and
HK entered the owering regulatory network through the sugar
signaling and hormone signaling pathways to regulate oral
transition in L. gratissima.
Phytohormones Regulate Floral Transition
in L. gratissima
Phytohormones play important regulatory roles in plant
development and the mechanisms of their participation in oral
transition in many plants are extensively studied (Shu et al.,
2018; Lin et al., 2019; Zhang et al., 2019; Bao et al., 2020).
However, the complex hormone regulatory network of oral
transition in perennial woody plants remains unclear. Westudied
the regulatory patterns of hormones that participate in oral
transition in L. gratissima.
As one of the most important phytohormones, the function
of GA in regulating oral transition is mainly achieved through
maintaining GA homeostasis and regulating the levels of DELLA,
a growth inhibitor in the GA signaling pathway (Bao et al.,
2020). GA homeostasis in plants is maintained through
coordinating the expression levels of the GA biosynthesis genes,
such as GA3OXs and GA20OXs, and the catabolic enzyme
genes GA2OXs, thereby regulating oral transition (Mateos
et al., 2015; Bao et al., 2020). In this study, homologs of
GA2OX1 (Unigene0030732) and GA2OX8 (Unigene0073113)
were both upregulated in SD10-vs.-LD10 (Figure 5C and
Supplementary Table S9). GA2OXs can catalyze the
2β-hydroxylation of bioactive GAs (such as GA1, GA3, GA4,
and GA9), resulting in decreased levels of bioactive GAs (Rieu
et al., 2008). is may be one of the reasons for low GA3
content in shoot apexes and leaves of L. gratissima. e main
components of GA signaling include the GA receptor GID1B
and the growth inhibitors, DELLAs (Bao et al., 2020). When
GA concentrations increase, the DELLA protein forms a
GA-GID1B-DELLA complex that undergoes degradation by
the ubiquitination pathway, thereby regulating the expression
of downstream genes (Bao et al., 2020). e GA signaling
pathway mainly promotes oral transition by inducing the
expression of SOC1 and LFY (Blázquez etal., 1998; Hou etal.,
2014; Bao et al., 2020; Fukazawa et al., 2021). In this study,
RGL3 (Unigene0071862) encoding DELLA had low expression
in SD10, SD13, and SD19 (Figure 5D and
Supplementary Table S9). In contrast, SOC1 (Unigene0039572
and Unigene0039575) and LFY (Unigene0030979) were highly
expressed in SD10, SD13, and SD19 (Figures 5G,H and
Supplementary Table S9). is showed that low expression
levels of the DELLA gene RGL3 could induce the expression
of SOC1 and LFY. Additionally, the GA receptor genes GID1Bs
(Unigene0032780, Unigene0032781, and Unigene0063035) were
upregulated in SD10-vs.-LD10 (Figure 5D and
Supplementary Table S9), further demonstrating that GA
promotes oral transition in L. gratissima. However, it may
not be GA1, GA3, GA4, or GA9 but other active GAs that
took eect. Previous studies indicated that GA has a promoting
eect in oral transition in A. thaliana (Yamaguchi etal., 2014;
Bao etal., 2020), whereas GA was found to negatively regulate
oral transition in woody plants (Li etal., 2018). GA regulation
of oral transition in L. gratissima (a woody plant) is similar
to herbaceous plants but not woody plants. is unique regulation
pattern may beaected by many endogenous and environmental
factors, which needs to be further studied in the future.
Other hormones also have some eects in regulating oral
transition in L. gratissima. ABA is usually considered a stress-
related hormone, but it also plays an important role in plant
development (Yoshida etal., 2019). However, there is still debate
over the role of ABA in oral transition because both promoting
and inhibitory eects were reported (Shu et al., 2018; Xiong
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 13 August 2021 | Volume 12 | Article 715683
et al., 2019). In this study, the ABA synthase gene NCED1
(Unigene0042754 and Unigene0042755) and the catabolic gene
CYP707A1 (Unigene0034382) were both upregulated in SD10-
vs.-LD10 (Figure 5C and Supplementary Table S9), and the
ABA content in the SAMs was maintained at high levels that
initially increased from SD0 to SD13 and subsequently declined,
reaching its peak on SD13 (Figure 3). ABF2 is a bZIP
transcription factor that binds to ABA. It is also an important
component of the glucose signaling pathway (Kim etal., 2004).
In this study, ABF2 (Unigene0046988) was highly expressed
in SD10, and likely participated in oral transition in L.gratissima
by mediating the ABA and glucose signaling pathways. In the
ABA core signaling pathway, the protein phosphatase PP2C
(ABI1, ABI2, HAB1, and PP2CA/AHG3) acts as a key negative
regulatory factor, which has important regulatory eects on
the activation of ABA signaling (Tischer et al., 2017). When
ABA levels increase in plants, the ABA receptors PYR1/PYLs/
RCARs bind and inhibit the phosphatase activity of PP2C,
thereby activating the ABA signaling pathway (Tischer et al.,
2017). In this study, PYL4 expression was high in SD13, whereas
PP2C expression peaked on SD10 but was also high on SD13
(Figure5D and Supplementary Table S9), suggesting that the
activation of the ABA signaling pathway mainly occurred on
SD13 and that ABA promoted ower development in L. gratissima
through the core signaling pathway. EDL3 is a positive regulator
of the ABA signal cascade reactions, and it positively regulates
the expression of the central component CONSTANS (CO) in
the photoperiod pathway to regulate oral transition (Koops
et al., 2011). In this study, the expression of EDL3 and COL12
in the photoperiodic pathway peaked on SD10 (Figures 5A,D
and Supplementary Table S9), suggesting that ABA promoted
oral transition in L. gratissima by interacting with EDL3 to
induce COL12 expression.
Plant growth depends on the continuous function of
meristems, and CKs have positive eects on SAMs. In this
study, the cytokinin synthase gene LOGs and the zeatin
O-glucosyltransferase gene ZOG1 were mainly upregulated in
SD10-vs.-LD10 and SD13-vs.-LD13 (Figures 5C,D and
Supplementary Table S9). It is known that zeatin O-glucoside
plays important roles in the transport and storage of CKs
(Kiran etal., 2012). On the other hand, the trans-zeatin synthase
gene CYP735A1 and the cytokinin oxidase/dehydrogenase gene
CKX7 were downregulated in SD19-vs.-LD19 (Figures 5C,D
and Supplementary Table S9). Zeatin promotes cell division
and has an important role in the early stages of ower bud
development and cell division. is is likely the reason zeatin
content gradually decreased from SD0 to SD19 (Figure 3).
e CK signaling pathway mainly cross talks with AGAMOUS
(AG) to regulate SAM dierentiation and maintenance (Zhang
et al., 2018). RPN12A participates in ATP-dependent
ubiquitinated protein degradation, which may inhibit the
degradation of one or more factors in CK signaling and balance
the proliferation rate of cells during bud development (Ryu
et al., 2009). In this study, AHPs, which are key components
in the cytokinin two-component signaling system (Liu et al.,
2017), were highly expressed mainly at SD10, SD13, and SD19;
ARR6, which is a CK responsive regulator (Liu et al., 2017),
was signicantly upregulated in SD19-vs.-LD19, and RPN12A
was upregulated in SD13-vs.-LD13; and moreover, AGL8 was
highly expressed in SD10, SD13, and SD19 (Figures 5D,H
and Supplementary Table S9), demonstrating that CK promotes
oral transition and ower development in L. gratissima indirectly
through the eects of AGL8.
In the JA signaling pathway, JAZ (jasmonate-ZIM domain,
TIFY family) and MYC2/3/4 regulate oral transition in plants
(Bao et al., 2020; Guan et al., 2021). In this study, TIFYs and
MYC4 were upregulated in SD10-vs.-LD10 (Figure 5D and
Supplementary Table S9), showing that the JA signaling pathway
promotes oral transition in L. gratissima. In SL signaling
pathway, D14 negatively regulates SL signals as an SL receptor
(Chevalier et al., 2014). In this study, D14 (Unigene0028658)
expression was high at the early stage of SD treatment, and
as treatment duration increased, its expression level decreased
(Figure 5D and Supplementary Table S9), which may have
been caused by negative feedback regulation of SL signals by
D14, thereby regulating SL changes during oral transition in
L. gratissima. CCD7 is a key enzyme in SL biosynthesis (Bao
et al., 2020). Compared with the LD treatment, CCD7
(Unigene0069349) expression was lower in response to SD
treatment and was signicantly downregulated in SD10-vs.-LD10
(Figure 5C and Supplementary Table S9), suggesting that SL
may inhibit oral transition in L. gratissima. In contrast to
the results of this study, recent studies have shown that SL
inhibits melatonin synthesis, thereby inducing oral transition
in A. thaliana in an FLC-dependent manner (Zhang et al.,
2019). As L. gratissima is a perennial woody plant, there may
be dierences in SL regulatory mechanisms in oral transition
compared with A. thaliana, which requires further
in-depth studies.
YUC-mediated auxin biosynthesis is vital for the formation
of oral organs (Cheng etal., 2006). In this study, YUC4 was
upregulated in SD19-vs.-LD19 (Figure 5C and
Supplementary Table S9), whereas IAA content increased from
SD10 to SD13 and continuously decreased aerward (Figure3),
whereas the auxin response gene SAUR7 was upregulated in
SD13-vs.-LD13 and SD19-vs.-LD19. ese results suggested
that auxin does not participate in regulating oral transition
in L. gratissima but instead has positive eects on the formation
of oral organs.
ese hormones interacted with other owering regulation
pathways to further ensure that L. gratissima rapidly responded
to changes in environmental and endogenous signals to precisely
regulate owering time.
Flowering Pathways During Floral
Transition in L. gratissima
e photoperiod pathway is involved in plant response to changes
to day length and circadian rhythm, making it one of the most
important owering regulation pathways. In the photoperiod
pathways of many plants, the bZIP transcription factor FD forms
a transient complex in SAMs with the FT protein from leaves
to induce the expression of oral meristem identity genes, thereby
promoting oral transition (Abe et al., 2019). In this study, FD,
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 14 August 2021 | Volume 12 | Article 715683
AP1, FUL, and AGL8 were highly expressed in SD10 and SD13
(Figures 5A,H), demonstrating that the FD protein directly or
indirectly induced the expressions of AP1, FUL, and AGL8,
thereby promoting oral transition in L. gratissima. CO is an
important regulatory factor in the photoperiod pathway, and
the expression of CO is regulated by a photoreceptor and circadian
rhythm in A. thaliana, and when the expression rhythm of CO
is consistent with the external photoperiod, expression of the
downstream gene SOC1 is activated (Goretti et al., 2020). In
this study, COL12 was upregulated in SD13-vs.-LD13 (Figure5A
and Supplementary Table S9), suggesting that the eects of
COL12 in ower development in L. gratissima were similar to
those of CO in A. thaliana.
e transcription factor LUX is one of the components of
evening complex (EC) in circadian rhythm and forms the HOS15-
EC-HDA9 histone-modifying complex in A. thaliana to inhibit
GI transcription, thereby inhibiting photoperiod-dependent
owering (Park etal., 2019). In this study, LUX was downregulated
in SD10-vs.-LD10 (Figure 5A and Supplementary Table S9),
indicating that LUX had inhibitory eects on oral transition
in L. gratissima. PRR7 positively regulates CO expression to
promote oral transition in long-day plants, whereas the PRR7/
PRR3 genes delay oral transition by inhibiting CO expression
in short-day plants (Nakamichi et al., 2020). In this study, PRR7
was downregulated in SD13-vs.-LD13 (Figure 5A and
Supplementary Table S9), showing that PRR7 inhibits oral
transition in L. gratissima, which was similar to the other
short-day plants. In A. thaliana, FKF1 could degrade CDF1
(factor inhibiting CO transcription) to regulate CO expression
and could directly bind to CO, or inhibit COP1 to stabilize
CO expression, thereby promoting owering (Lee et al., 2018).
However, FKF1 was downregulated in SD13-vs.-LD13 (Figure5A
and Supplementary Table S9), which was not consistent with
COL12 expression. is indicated that FKF1 inhibited oral
transition in L. gratissima and does not interact with COL12,
but other mechanisms may be present that require further study.
NF-Ys interact with CO in the photoperiod pathway to directly
regulate SOC1 transcription (Hou et al., 2014). In this study,
NF-Ys, COL12, and SOC1 were highly expressed in SD10 and
SD19 (Figures 5A,H), showing that NF-Ys may interact with
COL12 in the photoperiod pathway in L. gratissima to induce
SOC1 expression, thereby positively regulating oral transition
and owering development in L. gratissima.
Previous studies showed that ambient temperature-associated
EARLI1 regulated critical genes in the LD photoperiod pathway
in A. thaliana to promote FLC expression and delayed owering
time (Shi et al., 2011). In contrast, pEARLI1 was upregulated
in SD13-vs.-LD13 and SD19-vs.-LD19in this study (Figure5B
and Supplementary Table S9), indicating that pEARLI1 promoted
oral transition and ower development in L. gratissima.
In A. thaliana, age signals negatively regulate miR156 levels
to promote SPL accumulation (Yao et al., 2019). At SAMs,
SPLs target FUL and SOC1 or directly regulate AP1 transcription
to promote owering (Wang et al., 2009). In this study, SPL4
was upregulated in SD10-vs.-LD10, SD13-vs.-LD13, and SD19-
vs.-LD19 (Figure 5F and Supplementary Table S9), which
was consistent with the expression patterns of SOC1, FUL,
and AP1 (Figures 5G,H), indicating that the aging pathway
promoted oral transition and ower development in L. gratissima
through SPL4-induced expression of FUL, SOC1, and AP1.
e oral integrators SOC1 and AGL24 integrate various
owering signals from photoperiod, temperature, hormone, and
age-related signals to activate or inhibit downstream oral
meristem identity genes, and ultimately lead to the transformation
of vegetative to oral meristems in plants (Blümel etal., 2015).
SOC1 can be indirectly activated by CO (Lee and Lee, 2010).
At SAMs, when SOC1 is activated, SOC1 and AGL24 form
a heterodimer to directly activate LFY (Lee et al., 2008). In
this study, SOC1, AGL24, and LFY were highly expressed in
SD10, suggesting that SOC1 and AGL24 can jointly promote
LFY at this period to promote oral transition in L. gratissima.
During early ower development, AP1 activates A function
to inhibit SOC1 and AGL24 expression to prevent owering
reversion (Lee and Lee, 2010). In SD19, AGL24 and SOC1
expression decreased and AP1 expression increased
(Figures5G,H). ese changes may prevent dierentiated oral
meristems from undergoing owering reversion.
SEPs are important regulatory factors during ower development
and form a heterodimer with AP1 to regulate genes during oral
meristem development (Jetha et al., 2014). In this study, SEPs
were highly expressed in SD10, SD13, and SD9, which was
consistent with AP1 expression (Figure 5H), showing that AP1
mediated positive regulation of oral transition and early ower
development in L. gratissima by SEPs. In Arabidopsis, SVP is a
owering inhibitor and plays a role in oral transition by directly
inhibiting SOC1 expression at SAMs and leaves (Li etal., 2008).
In this study, SVP had low expressions in SD10, SD13, and
SD19, whereas SOC1 expression was high (Figures 5G,H),
indicating that low levels of SVP induced SOC1 expression to
promote oral transition and ower formation in L. gratissima.
TFL1 is a key regulatory factor of oral transition and
inorescence meristem development in A. thaliana. TFL1 and
FT have highly conserved amino acid sequences but opposite
gene functions: FT promotes owering, whereas TFL1 inhibits
owering (Jin etal., 2020). Previous studies showed that TFL1
negatively regulated transcription of the target gene FD, thereby
regulating the owering time and inorescence meristem
development (Hanano and Goto, 2011). In this study, TFL1
had low expression at SD10 and SD13, which is the opposite
of FD expression (Figures 5A,H), indicating that low levels
of TFL1 promoted FD expression and, therefore, oral transition
in L. gratissima.
Figure 6 shows the hypothetical model of the regulatory
network of SD photoperiod-induced oral transition in L. gratissima,
involved in the regulation of multiple owering signals in oral
transition, including signals for photoperiod, phytohormones (GA,
ABA, CK, JA, and SL), sugar, ambient temperature, age, and
oral integrator and oral meristem identity genes.
CONCLUSION
Our study enables a comprehensive understanding of the gene
expression patterns occurring during SD photoperiod-induced
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 15 August 2021 | Volume 12 | Article 715683
oral transition in L. gratissima. e histological, endogenous
substance contents, and dierential gene expression analyzes
showed that short-day photoperiod activated systemic responses
in L. gratissima and induced the generation of owering signals
in the photoperiod pathway. Furthermore, a complex regulatory
network, including GA, ABA, CK, JA, and SL signals, sugar
signals, and temperature and age signals, was formed through
the integration of SOC1 and AGL24. e outcomes of this
study will aid in understanding owering time regulation in
L. gratissima at the molecular level, provide theoretical guidance
for achieving year-round production, and further provide a
reference for understanding the regulatory mechanisms of
owering time in other woody plants.
DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the
article/Supplementary Material. e raw data of RNA sequencing
from this study have been deposited into the NCBI Sequence
Read Archive (SRA) database (BioProject ID: PRJNA648802).
e de novo transcriptome has been deposited at GenBank
under the accession GIXA00000000.
AUTHOR CONTRIBUTIONS
ZL, YW, and HM conceived and designed the study and revised
the manuscript. XioL, YW, JA, XZ, YC, and XiuL conducted the
experiment and collected the plant materials. XioL and YW analyzed
and interpreted the data. XioL and YW wrote the manuscript.
All authors read and approved the nal version of the manuscript.
FUNDING
is work was funded by the Fundamental Research Funds
for the Central Non-prot Research Institution of CAF
(CAFYBB2019ZB007 to ZL), the Fundamental Research Funds
for the Central Non-prot Research Institution of CAF
(CAFYBB2017MB014 to YW), and Ten ousand Talent Program
of Yunnan Province (grant to HM).
FIGURE6 | Proposed regulatory network of short-day photoperiod-induced oral transition in L. gratissima. Colored fonts represent downregulated (green) or
upregulated (red) genes.
Liu et al. Photoperiod-Induced Floral Transition of Luculia gratissima
Frontiers in Plant Science | www.frontiersin.org 16 August 2021 | Volume 12 | Article 715683
ACKNOWLEDGMENTS
e authors would like to thank Guangzhou Genedenovo
Biotechnology Co., Ltd. for assisting in RNA
sequencing.
SUPPLEMENTARY MATERIAL
e Supplementary Material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fpls.2021.715683/
full#supplementary-material
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Frontiers in Plant Science | www.frontiersin.org 19 August 2021 | Volume 12 | Article 715683
GLOSSARY
Term Denition
ABA abscisic acid
AG AGAMOUS
AGL24 AGAMOUSLIKE24
AP1 APETALA1
BGLU24 BETA-GLUCOSIDASE 24
CAB40 CHLOROPHYLL A-B BINDING PROTEIN
CCD7 CAROTENOID CLEAVAGE DIOXYGENASE 7
CK cytokinin
CO CONSTANS
COL12 CONSTANS-LIKE 12
CYP734A1 CYTOCHROME P450 734A1
D14 DWARF14
DEG differentially expressed gene
EC evening complex
EDL3 EID1-LIKE F-BOX PROTEIN 3
FKF1 FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1
FT FLOWERING LOCUS T
FUL FRUITFULL
GA gibberellin
GA2OX1 GIBBERELLIN 2-BETA-DIOXYGENASE 1
GOLS2 GALACTINOL SYNTHASE 2
HK HEXOKINASE
HPLC-MS high-performance liquid chromatography-mass spectrometry
HSFs HEAT STRESS TRANSCRIPTION FACTORS
HSPs HEAT SHOCK PROTEIN/COGNATE
IAA indole-3-acetic acid
JA jasmonic acid
JMT JASMONATE O-METHYLTRANSFERASE
LD long day
LFY LEAFY
NF-Y nuclear factor Y
PRR7 PSEUDO-RESPONSE REGULATOR 7
RFS RAFFINOSE SYNTHASE
SD short day
SEP3 SEPALLATA3
SEPs SEPALLATAs
SL strigolactone
SOC1 SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1
SPL4 SQUAMOSA PROMOTER-BINDING-LIKE PROTEIN 4
SVP SHORT VEGETATIVE PHASE
T6P trehalose-6-phosphate
TFL1 TERMINAL FLOWER 1
TPS TREHALOSE-6-PHOSPHATE SYNTHSE
UGT74F1 UDP-GLYCOSYLTRANSFERASE 74F1
WGCNA weighted gene co-expression network analysis
ZT zeatin
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