Abstract
Liquidambar formosana Hance has a highly ornamental value as an
important urban greening tree species with bright and beautiful leaf
color. To gain insights into the physiological and molecular mechanisms
of L. formosana leaf color change, the leaves of three different clones
were sampled every ten days from October 13, 2019, five times in total,
which are S1, S2, S3, S4 and S5. Transcriptome sequencing was performed
at S1 and S4. The chlorophyll content of the three clones decreased
significantly, while the anthocyanins content of the three clones
increased significantly in the coloring stage. The anthocyanins content
of clone 2 was far more than that of the other two clones throughout
the period of leaf color change. The transcriptome analysis showed that
six DEGs related to anthocyanins biosynthesis, including CHS (chalcone
synthase), CHI (chalcone isomerase), F3′H (flavonoid 3′-hydroxylase),
DFR (dihydroflavonol 4-reductase), ANS (anthocyanidin synthase) and FLS
(flavonol synthase), were found in three clones. Clone 2 has another
three DEGs related to anthocyanins biosynthesis, including PAL
(Phenylalanine ammonia-lyase), F3′5′H (flavonoid 3′,5′-hydroxylase) and
UFGT (flavonoid 3-O-glucosyltransferase). We lay a foundation for
understanding the molecular regulation mechanism of the formation of
leaf color by exploring valuable genes, which is helpful for L.
formosana breeding.
Keywords: Liquidambar formosana, anthocyanins biosynthesis pathway,
RNA-Seq, DEGs, leaf coloration
1. Introduction
Liquidambar formosana Hance, a deciduous ornamental tree species, is
widely distributed in Southeast Asia [[38]1,[39]2]. This plant is one
of the main afforestation tree species in southern China, which
exhibits strong adaptability, fire resistance and high ecological
benefits [[40]3]. L. formosana has great potential in landscaping and
gardening, owing to the fact that its leaf color changes seasonally
from green to yellow or red in October [[41]4]; therefore, studying the
leaf color of L. formosana is of great importance for breeding
individual clones with good ornamental values.
Autumn leaf coloration is one of the most important horticultural
characteristics in nature [[42]5]. Various colored-leaf trees have
gradually become the primary choice for landscaping due to their
colorful leaves improving the level and ornamental value of the
landscape [[43]6]. Leaf color is affected by the accumulation of
different types and quantities of pigments, such as flavonoids and
anthocyanins [[44]7]. Flavonoids are able to regulate pigmentation and
photo-protection [[45]8]. Anthocyanins are an important class of
flavonoids that represent a large group of plant secondary metabolites.
They are glycosylated polyphenolic compounds with a range of colors
varying from orange, red and purple to blue in flowers, seeds, fruits
and vegetative tissues [[46]9]. Anthocyanins are involved in defense
responses against pathogens, protecting plants from strong light and UV
radiation [[47]10]. They have antioxidant properties to protect plants
against various biotic and abiotic stresses [[48]11]. In addition,
anthocyanins accumulate in the flowers and fruits of plants and are
responsible for their rich color attracting pollinators and seed
dispersers [[49]12]. They were catalyzed by complex enzymes from
phenylpropanoid and flavonoid biosynthetic pathways [[50]13]. A wide
range of constructive genes was involved in the anthocyanin’s
biosynthesis. In Arabidopsis thaliana, the structural enzymes in the
anthocyanins biosynthetic pathway include the upstream components
chalcone synthase (CHS), chalcone isomerase (CHI) and the downstream
components dihydroflavonol 4-reductase (DFR), anthocyanidin synthase
(ANS) and others [[51]14]. The structural genes involved in
anthocyanins biosynthesis have been identified in genetic analyses of
some plants, such as cucumber and sainfoin flowers [[52]15,[53]16]. To
date, little is known about the molecular regulatory mechanism of the
key genes underlying leaf color change in L. formosana. Many studies on
L. formosana have explored the relationship between leaf color change
and environmental factors by measuring the content changes of
chlorophyll and other related pigments. Studies of Hu and Liu have
pointed out that temperature is the main factor affecting the
accumulation of pigment in L. formosana leaves [[54]17,[55]18]. This
study can enrich the knowledge about the molecular mechanisms of L.
formosana.
Understanding of the metabolic pathways involved in leaf color change
during L. formosana development requires the exploration of functional
genes. RNA sequencing, based on deep sequencing, has been widely used
for gene discovery and analysis of specific genes [[56]19]. The results
of this study not only accessed key genes in the anthocyanins
biosynthetic pathway of three clones of L. formosana in autumn, but
also aimed to explore the molecular mechanism of its color formation,
thereby providing a theoretical basis for L. formosana molecular
breeding.
2. Results
2.1. Dynamic Patterns of Chlorophyll, Carotenoid and Anthocyanin Content
The total chlorophyll, carotenoid and anthocyanins in leaves varied
significantly at different coloration stages ([57]Figure 1). With
increased redness in leaf color, the total chlorophyll levels decreased
([58]Figure 1a), while anthocyanin levels increased ([59]Figure 1c) and
carotenoid levels fluctuated ([60]Figure 1b). For the total
chlorophyll, the contents of the three clones at the S1 stage were 2.64
mg/g, 2.87 mg/g and 2.96 mg/g, respectively ([61]Figure 1a), while the
contents at the S4 stage were 1.78 mg/g, 1.82 mg/g and 0.52 mg/g,
respectively. When compared with S1, the total chlorophyll decreased by
32.6%, 36.6% and 82.4%, respectively. For carotenoids ([62]Figure 1b),
the contents of the three clones at the S1 stage were 0.17 mg/g, 0.14
mg/g and 0.09 mg/g, respectively, while content at the S4 stage were
0.02 mg/g, 0.08 mg/g and 0.05 mg/g, respectively. When compared with
S1, carotenoids decreased by 88.2%, 42.8% and 44.4%, respectively. For
anthocyanins ([63]Figure 1c), levels of the three clones at the S1
stage were 10.75 U/g, 18.20 U/g and 15.57 U/g, respectively, while
levels at S4 stage were 24.34 U/g, 61.59 U/g and 20.14 U/g,
respectively. Compared with S1, anthocyanins increased by 2.26, 3.38
and 1.29 times. In addition, the anthocyanins levels of clone 2 were
far more than that of the other two clones throughout the period of
leaf color change.
Figure 1.
[64]Figure 1
[65]Open in a new tab
Changes in total chlorophyll, carotenoids and anthocyanin contents
during the leaf coloration: (a) changes in total chlorophyll content in
different clones; (b) changes in carotenoids content in different
clones; (c) changes in anthocyanin content in different clones.
Different lowercase letters within each graph indicate significant
differences (p < 0.05) among different clones, while different capital
letters within each graph indicate significant differences among
different time periods (p < 0.05). There were three biological
replicates in each clone at each stage.
2.2. De Novo Assembly of L. formosana Transcriptome
To explore changes in transcription, a total of 18 samples of three
clones at S1 and S4 stages of L. formosana were selected, consisting of
three clones with three replicates per clone. A total of 936,385,220
raw reads with a total of 141,394,168,220 bp were obtained. After
removing the adaptor and low-quality reads, a total of 927,523,280
clean reads with a total of 136,254,863,719 bp were obtained from the
eighteen sequencing libraries for further analysis ([66]Table S1).
2.3. Gene Annotation and Classification
In total, 196,890 unigenes (99.65% of the 197,577 total unigenes) were
identified by BLASTx (E-value < 1 × 10^−5) in at least one of the GO,
COG and KEGG databases. In total, there were 82,229 (41.62%), 88,492
(44.79%) and 55,770 (28.23%) unigenes that had annotated to the GO, COG
and KEGG databases, respectively.
To analyze the functions of DEGs, we used the GO annotation term to
enrich the DEGs between S1 and S4 of the three clones. DEGs were
divided into three ontologies: molecular function, cellular component
and biological process ([67]Figure S1). In clone 1, for the molecular
function, “binding” and “catalytic activity” were the frequent terms
and were associated with 7577 and 7381 DEGs, respectively. For the
cellular component, the DEGs were mainly enriched for “cell part”,
“membrane part” and “organelle” with 6809, 4891 and 3892 DEGs,
respectively. For the biological process, the DEGs were mainly enriched
for “cellular process” (6526 DEGs) and “metabolic process” (6055 DEGs).
Similarly, in clone 2, for the molecular function, the DEGs were
associated with the “binding” (6758 DEGs) and “catalytic activity”
(6709 DEGs); for the cellular component ontology, the DEGs were also
enriched for genes involved in “cell part” (5692 DEGs), “membrane part”
(4401 DEGs) and “organelle” (3191 DEGs); for the biological process
ontology, the DEGs were mainly enriched for “cellular process” (5586
DEGs) and “metabolic process” (5173 DEGs). In clone 3, for the
molecular function, the DEGs were associated with the “binding” (6621
DEGs) and “catalytic activity” (6090 DEGs); for the cellular component
ontology, the DEGs were also enriched for genes involved in “cell part”
(4718 DEGs), “membrane part” (4335 DEGs) and “organelle” (2621 DEGs);
for the biological process ontology, the DEGs were mainly enriched for
“cellular process” (4852 DEGs) and “metabolic process” (4357 DEGs).
A total of 88,492 unigenes were assigned to 23 COG classifications,
with the majority (44,335, 50.10%) in “Function unknown”, followed by
“Translation, ribosomal structure and biogenesis” (7038, 7.95%) and
“Posttranslational modification, protein turnover, chaperones” (6378,
7.21%) ([68]Figure S2).
A total of 55,770 unigenes were assigned to six KEGG categories and 20
sub-categories ([69]Figure 2). “Metabolism” accounted for the highest
proportion, most of which were involved in “Carbohydrate metabolism
(6019, 10.79%) and “Amino acid metabolism” (3729, 6.69%). In “Genetic
information processing”, “Translation” had the highest number of
unigenes (8801, 15.78%), followed by “Folding, sorting and degradation”
(3939, 7.06%). In addition, “Transport and catabolism” had a high
proportion (3360, 6.02%).
Figure 2.
[70]Figure 2
[71]Open in a new tab
KEGG pathway classification statistics.
2.4. Analysis of DEGs
To analyze the dynamic expression patterns of the specific genes in
leaf color change, the differences in transcriptome profiles between
the S1 and S4 stages of different clones were compared. The Transcripts
Per Million (TPM) values were statistically analyzed to select
different unigenes by using the DESeq method [[72]20] ([73]Table S2).
We compared the DEGs of the three clones (S1-vs-S4). In clone 1, we
identified 20,020 DEGs with 16,005 upregulated and 4015 downregulated
([74]Figure 3a,b). In clone 2, there were 17,000 DEGs with 12,941
upregulated and 4059 downregulated ([75]Figure 3a,c). Similarly, there
are 17,397 DEGs in clone 3, of which 11,418 DEGs were upregulated and
5979 DEGs were downregulated ([76]Figure 3a,d). Based on these
analyses, the DEGs were found and significantly expressed in all three
clones, while some of DEGs in clone 2 were the most significant
([77]Figure 3c).
Figure 3.
[78]Figure 3
[79]Open in a new tab
(a) Differences in expression levels of different clones (S1-vs-S4);
(b) differences in expression levels of clone 1 (S1-vs-S4); (c)
differences in expression levels of clone 2 (S1-vs-S4); (d) differences
in expression levels of clone 3 (S1-vs-S4). In the volcano map, y-axis
represents the fold change value of gene expression difference between
two stages and x-axis shows the statistical test value of gene
expression difference; that is, the higher the point is, the more
significant the difference is; the farther away from the center, the
greater the multiple of difference.
2.5. KEGG Pathway Enrichment Analysis of DEGs
In this study, we carried out an enrichment analysis based on the KEGG
database in order to explore the biological functions of these DEGs. A
total of 8988 unigenes were assigned to 134 KEGG pathways in clone 1
between S1 and S4 stages. Similarly, a total of 7647 unigenes were
assigned to 122 KEGG pathways in the clone 2 (S1-VS-S4) and a total of
6341 unigenes were assigned to 135 KEGG pathways in the clone 3
(S1-VS-S4).
Most of the pathways annotated by the three clones were related to
“Metabolism”. In the pathways related to leaf color change, the three
clones were significantly enriched in “Phenylpropanoid biosynthesis”,
“Flavonoids biosynthesis”, “Isoflavonoid biosynthesis”, “Anthocyanin
biosynthesis”, “Phenylalanine metabolism” and other pathways
([80]Figure 4).
Figure 4.
[81]Figure 4
[82]Open in a new tab
Enrichment of differential genes KEGG in different clones: (a)
enrichment of differential genes KEGG in clone 1; (b) enrichment of
differential genes KEGG in clone 2; (c) enrichment of differential
genes KEGG in clone 3.
2.6. Identification of DEGs Related to Anthocyanin Metabolism
Based on annotations in public databases, a total of nine
anthocyanin-related genes with significant differential expression were
obtained, among which six genes were significantly upregulated in three
clones ([83]Table 1). TRINITY_DN11132_c0_g1, TRINITY_DN4277_c0_g1,
TRINITY_DN18660_c0_g2, TRINITY_DN11660_c0_g1, TRINITY_DN29005_c0_g1;
TRINITY_DN17255_c0_g3 were annotated as CHS, CHI, F3′H, DFR, ANS and
FLS, respectively. Another three genes, including
PAL(TRINITY_DN17802_c0_g4), F3′5′H(TRINITY_DN28662_c0_g1) and
UFGT(TRINITY_DN3115_c0_g1), were significantly upregulated in clone 2
([84]Table 2), indicating that there were more DEGs related to
anthocyanin biosynthesis in clone 2.
Table 1.
DEGs related to anthocyanin biosynthesis in three clones.
Gene ID Abbreviation Up/Down TPM Value
S1Clone1 S1Clone2 S1Clone3 S4Clone1 S4Clone2 S4Clone3
TRINITY_DN11132_c0_g1 CHS up 5501.80 411.20 420.20 11901.80 3553.40
3352.30
TRINITY_DN4277_c0_g1 CHI up 186.20 198.20 210.0 193.2 439.0 468.6
TRINITY_DN18660_c0_g2 F3′H up 286.40 3634.70 4101.80 944.1 16367.90
14578.70
TRINITY_DN11660_c0_g1 DFR up 3245.80 3301.50 2578.90 802.4 1000.0
1092.40
TRINITY_DN29005_c0_g1 ANS up 27.2 19.8 22.4 487.9 868.6 782.4
TRINITY_DN17255_c0_g3 FLS up 13.1 23.4 45.0 26.9 128.2 93.9
[85]Open in a new tab
Table 2.
DEGs related to anthocyanin biosynthesis in clone 2.
Gene ID Abbreviation Up/Down TPM Value
S1Clone2 S4Clone2
TRINITY_DN17802_c0_g4 PAL up 93.3 216.5
TRINITY_DN28662_c0_g1 F3′5′H up 1.8 94.5
TRINITY_DN3115_c0_g1 UFGT up 168.0 468.3
[86]Open in a new tab
To further validate the reliability of the RNA-seq results, eight DEGs
related to anthocyanin biosynthesis were selected ([87]Figure 5). The
relative expression of these key genes was very similar to the RNA-seq
results, suggesting that the RNA-seq data and DEG analysis are
reliable.
Figure 5.
[88]Figure 5
[89]Open in a new tab
Expression of leaf color-related unigenes of L. formosana quantified by
qRT-PCR. Bars with different lowercase letters are significantly
different (p < 0.05). There were three biological replicates in each
clone at each stage.
3. Discussion
3.1. Physiological Mechanism of Leaf Color Change of L. formosana
Seasonal changes in the content of chlorophyll, carotenoids and
anthocyanins in higher plants are the main reasons for the changes in
leaf color [[90]21]. The main components of chlorophyll are Chl A and
Chl B. Chl B is unstable and easily decomposed at low temperatures
[[91]18]. Anthocyanins are a general term for a large class of
compounds, which are flavonoids [[92]13]. Plant leaf color is closely
related to anthocyanins [[93]22]. Under acidic soil conditions,
anthocyanins appear red, and under alkaline soil conditions,
anthocyanins appear blue [[94]18]. In our study, the total chlorophyll
content of all three clones significantly decreased, but the
anthocyanin content increased from S1 stage to S4 stage. A similar
phenomenon was found in the study by Wen and Chu [[95]5]. In their
study, the content of chlorophyll decreased, and the content of
anthocyanin increased during the color change of L. formosana. Nie et
al. reached the same conclusion in the change of leaves of Cotinus
coggygria in autumn [[96]23] Tao et al. also showed that the change of
leaf color of poplar had the same findings [[97]24]. In addition, it
was found that clone 2 had the brightest color and the highest
anthocyanin content ([98]Figure 1c), which was consistent with the most
increased anthocyanin in the “red type” in Wen and Chu’s study [[99]5].
The increase in anthocyanins was the primary reason for the change in
leaf color in L. formosana, causing the brighter color leaves after the
coloration period [[100]25].
However, our study on carotenoids was slightly different from that of
Hu et al. [[101]17], who reported that the carotenoid content of L.
formosana leaves decreased significantly at first but did not change
significantly thereafter. The dynamic pattern of carotenoid content
during leaf color change of L. formosana needs further study.
3.2. Genes Involved in the Anthocyanin Biosynthesis Pathway
Anthocyanins are an important class of flavonoids that are widely
present in plants [[102]13]. Anthocyanin synthesis is catalyzed by a
series of enzymes in the phenylpropanoid and flavonoid pathways
[[103]26] ([104]Figure 6). The biosynthesis of anthocyanins is
controlled by structural genes and regulatory genes, of which
structural genes encode biosynthetic enzymes and play a catalytic role
in anthocyanin synthesis [[105]27].
Figure 6.
[106]Figure 6
[107]Open in a new tab
Schematic representation of the anthocyanin biosynthetic pathway. PAL,
phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL,
4-coumaroyl-coA synthase; CHS, chalcone synthase; CHI, chalcone
isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid
3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; DFR,
dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; UFGT,
flavonoid 3-O-glucosyltransferase; FLS, flavonol synthase. The “*”
means multiplication.
Phenylalanine ammonia-lyase (PAL) is an enzyme that catalyzes the first
step of the phenylpropanoid metabolic pathway [[108]28]. As early as
1960, Neish confirmed that PAL catalyzed the synthesis of anthocyanin
[[109]29]. PAL catalyzes the conversion of phenylalanine to cinnamic
acid, while C4H catalyzes the conversion of cinnamic acid to 4-coumaric
acid. The conversion of 4-coumaric acid to 4-coumaroyl-CoA is catalyzed
by 4CL [[110]26]. 4-Coumaroyl-CoA can generate anthocyanins through
flavonoid metabolism, which are important components of flower, fruit
and leaf color in plants. Moreover, the synthesis of these substances
is closely related to PAL activity [[111]30,[112]31,[113]32]. In our
study, one PAL (TRINITY_DN17802_c0_g4) gene in clone 2 was
significantly upregulated at S4 stage, and the expression pattern was
confirmed in the qRT-PCR analyses; however, we did not find any DEGs
annotated as C4H and 4CL, which encode the enzymes required for the
production of 4-coumaroyl-CoA.
The first committed enzyme in the flavonoid pathway is Chalcone
synthase (CHS), a polyketide synthase, mediating the synthesis of
naringenin chalcone from 4-coumaroyl-CoA and malonyl-CoA [[114]13].
Then, naringenin chalcone is isomerized by chalcone isomerase (CHI) to
naringenin, the direct precursor of all flavonoid substances [[115]8].
CHS is an important regulatory gene located upstream in the flavonoid
biosynthesis pathway, and its overexpression may positively affect the
expression of downstream chalcone isomerase (CHI) genes that affect the
production of flavonoids [[116]33,[117]34]. In our study, both CHS
(TRINITY_DN11132_c0_g1) and CHI (TRINITY_DN4277_c0_g1) gene showed
significantly higher expression levels in three clones at the S4 stage.
This result indicated that CHS does positively regulate CHI expression
during the leaf color change of L. formosana. At the same time, we also
found a significantly upregulated flavonol synthase (FLS) gene
(TRINITY_DN17255_c0_g3) in three clones, which encodes the enzyme that
catalyzes naringenin chalcone to flavonols [[118]30]. Flavanone
3-hydroxylase (F3H), which belongs to the OGD family, converts
naringenin into dihydrokaempferol that can be further hydroxylated by
flavonoid 3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H)
into two other dihydroflavonols, dihydroquercitin or dihydromyricetin,
respectively [[119]35,[120]36]. F3′H and F3′5′H are the key enzymes
determining the structures of anthocyanins, and therefore, they affect
color formation [[121]37]. In this study, none of the DEGs were
annotated as F3H; however, the DEG TRINITY_DN18660_c0_g2, which was
annotated as F3′H, showed a significantly higher expression level at
the S4 stage than at the S1 stage in three clones. Another upregulated
F3′5′H gene (TRINITY_DN28662_c0_g1) was screened from clone 2. This
result was similar to a study that determined the pathway by which red
longan (Dimocarpus longan) fruits were produced. Yi et al. revealed
that genes related to enzymes leading up to dihydromyricetin were
significantly upregulated in red pericarp longan fruits [[122]38]. This
may be the reason for the change in leaf color in L. formosana and may
also be responsible for the bright red color of clone 2 in autumn.
Next, the three dihydroflavonols are reduced to colorless
leucoanthocyanidins by dihydroflavonol 4-reductase (DFR). Anthocyanidin
synthase (ANS), which belongs to the OGD family, catalyzes the
synthesis of corresponding colored anthocyanidins [[123]39]. Nakatsuka
et al. showed that ANS gene mutations could cause gentian flowers to
turn white [[124]40]. In this study, two upregulated genes, DFR
(TRINITY_DN11660_c0_g1) and ANS (TRINITY_DN29005_c0_g1) genes were
excavated, and their expression levels increased significantly in all
three clones at the S4 stage. In the end, anthocyanidins are decorated
and glycosylated by various members of the glycosyltransferase enzyme
family, for instance, flavonoid 3-O-glucosyltransferase (UFGT)
[[125]9]. We found that the transcript level of UFGT
(TRINITY_DN3115_c0_g1) in clone 2 was higher at the S4 stage than at
the S1 stage. This result indicated that the biosynthesis of
anthocyanin compounds is maintained at high levels in clone 2 at the S4
stage. The higher expression levels of PAL, CHS, CHI, F3′H, F3′5′H,
DFR, ANS, UFGT and FLS in red leaf than in green leaf of L. formosana
suggested that these genes are responsible for leaf color formation.
4. Materials and Methods
4.1. Plant Materials and Growth Conditions
The clones were selected; the trees originated from Qimen county, Anhui
province and were vegetatively propagated by grafting. They were
planted in the Practice Forest Farm of Nanjing Forestry University
(located in XiaShu town, JuRong County, Jiangsu province 32°07′ N,
119°13′ E). In mid-October, 2019, three clones with the most
representative coloration effects were selected for sampling. Beginning
October 13th, fresh leaves were collected approximately every ten days.
The leaves were immediately frozen in liquid nitrogen and stored at −80
°C until use. The five developmental stages were defined according to
the time of collection: S1, the green leaf stage; S2, leaf with red
margin stage; S3, leaf with red range-expanding stage; S4, the red leaf
stage; S5, the red faded stage ([126]Figure 7).
Figure 7.
[127]Figure 7
[128]Open in a new tab
Different leaf colors of L. formosana at the five developmental stages.
4.2. Analysis of Pigment Content
Chlorophyll and carotenoid content of leaves from three clones were
measured on the basis of the procedure described by Lichtenthaler and
Wellburn [[129]41]. Approximately 0.2 g of fresh samples were ground
until no visible tissue in 2 mL of 95% ethanol with a small amount of
quartz sand. Then add another 10 mL of 95% ethanol, grind into a
homogenate, filter and dilute to 25 mL with ethanol. Finally, the
chlorophyll extract was measured by spectrophotometer at the absorption
wavelengths of 665 nm, 649 nm and 470 nm. The measurements were
performed with three biological replicates.
[MATH: Chlorophyll A concentration (mg/L)=13.95O
D665−
6.88OD649
:MATH]
(1)
[MATH: Chlorophyll B concentration (mg/L)=24.96O
D649−
7.32OD665
:MATH]
(2)
Total chlorophyll concentration (mg/L) = Chl A + Chl B (3)
[MATH:
Carotenoid concentration Car (mg/L)=(1000<
mi>OD470−
2.05 Chl A−114.8
Chl B)/245 :MATH]
(4)
Pigment content (mg/g) = (pigment concentration ∗ extraction liquid
volume ∗ dilution ratio)/sample fresh weight (5)
The content of anthocyanins was determined by the hydrochloric
acid-ethanol extraction method [[130]15,[131]42]. In total, 3 g samples
were taken and divided into three parts. Anthocyanins were extracted in
10 mL ethanol (containing 1% hydrochloric acid) for 4 h at 32 °C in
darkness. The samples were centrifuged at 5000 RPM for 10 min.
Supernatants were taken to measure the absorbance at 520 nm, which is
the absorbance OD value, repeated three times. Taking 0.1 OD value of
fresh weight per gram of leaves in 10 mL extract as a pigment range U,
the relative content of anthocyanin is as follows:
[MATH:
Anthocyanin content
(U/g)=OD520/0.1
:MATH]
(6)
4.3. RNA Extraction, cDNA Library Construction and Sequencing
The L. formosana leaves at two stages (S1 and S4) were selected as
materials. The leaves of S1 are the green ones before coloration, and
the leaves of S4 are the red ones after coloration. There were three
clones per stage, and each clone had three biological replicates,
resulting in a total of 18 samples. Total RNA was extracted using the
Plant RNA Kit (Omega Bio-Tek, Doraville, GA, USA) according to the
manufacturer’s instructions. The quantity and quality of total RNA were
assessed using a 1% agarose gel and a Nanodrop ND 2000
spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Total
RNA integrity and concentration were assessed using the Bioanalyzer
2100 RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA).
Poly(A) mRNA was isolated from total RNA using the Oligotex mRNA Mini
Kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer’s
instructions. The cDNA library was built using methods previously
described by Niu et al. [[132]43]. The 18 cDNA libraries were sequenced
on the Illumina Hiseq 4000 Sequencing platform (Illumina, Inc., San
Diego, CA, USA). The raw data were processed to remove low-quality
sequences (more than 50% of reads with Q < 19 bases),
adapter-contaminated sequences, and sequences with more than 5%
ambiguous base sequences. Clean reads were assembled into unigenes by
Trinity software (Trinity Release v2.4.0, MIT and Harvard, Cambridge,
MA, USA) [[133]44].
4.4. Unigene Annotation and DEG Analysis
Assembled unigenes were aligned to publicly available protein
databases, including GO(GeneOntology, [134]http://www.geneontology.org,
accessed on 3 April 2022), COG(Clusters of Orthologous Groups of
proteins, [135]http://www.ncbi.nlm.nih.gov/COG/, accessed on 3 April
2022) and KEGG(Kyoto Encyclopedia of Genes and Genomes,
[136]http://www.genome.jp/kegg/, accessed on 3 April 2022).
Unigenes expression was normalized to Transcripts Per Million (TPM) and
the DEGs between different stages were identified with padj < 0.05 and
|log2 (foldchange value)| ≥ 1 [[137]45] Next, GO and KEGG enrichment
analysis was performed on all DEGs, and a hypergeometric test with a
threshold of p ≤ 0.05 determined significant enrichment of GO terms and
KEGG pathways.
4.5. qRT-PCR Validation
Eight key genes involved in anthocyanin biosynthesis were selected for
validation by quantitative real-time PCR (QRT-PCR). The primers were
designed by Primer Premier 5.0 (Premier Biosoft International, Palo
Alto, CA, USA) and the reference gene was 18S ribosomal RNA [[138]46].
All experiments were performed using the StepOne Real-Time PCR System
(Applied Biosystems, Foster City, CA, USA) using SYBR Green Dye
(Takara, Dalian, China). DEGs were analyzed using the
[MATH: 2−ΔΔCt<
/mrow> :MATH]
method [[139]47]. The experiment was conducted with three biological
replicates, and each biological replicates had three technical
replicates. The gene-specific primers designed for nine candidate DEGs
are listed in [140]Table S3.
4.6. Statistical Analysis
The data analysis included a basic descriptive analysis followed by an
analysis of variance (ANOVA). Significant differences were based on
Duncan’s test, which were performed using SPSS 23.0 for Windows (SPSS
Science, Chicago, IL, USA). The p-values less than 0.05 were considered
to indicate significance between groups. For the elaboration of graphs,
Excel 2019 (Microsoft, Redmond, WA, USA) was used.
5. Conclusions
Overall, the regulation mechanism of leaf color in L. formosana was
firstly carried out by physiology and RNA-seq. It was found that with
increased redness in leaf color, the total chlorophyll levels
decreased, while anthocyanin levels increased. The anthocyanins content
of clone 2 was far more than that of the other two clones throughout
the color-changing period. Six genes, including CHS, CHI, F3′H, DFR,
ANS and FLS, play an important role in the anthocyanin’s biosynthesis
pathway in three clones. Another three genes, including PAL, F3′5′H and
UFGT, were only significantly expressed in clone 2, indicating that
there were more DEGs related to anthocyanin biosynthesis in clone 2.
Our study will provide molecular information for the selection and
breeding of new species of colored-leaf species and provide a reference
for the future study of leaf color polymorphisms in L. formosana.
Supplementary Materials
The following supporting information can be downloaded at:
[141]https://www.mdpi.com/article/10.3390/molecules27175433/s1, Figure
S1: GO annotations analysis; Figure S2: COG classification statistics;
Table S1: Transcriptome sequencing data; Table S2: Statistical analysis
of TPM value; Table S3: Primers used in qPCR.
[142]Click here for additional data file.^ (6.2MB, zip)
Author Contributions
Conceptualization, F.Y.; methodology, Y.Z. and F.Y.; validation, Y.L.,
H.C. and C.C.; formal analysis, Y.Z. and Y.L.; investigation, Y.L. and
Z.L.; resources, Y.Z and C.H.; data curation, Q.W.; writing—original
draft preparation, Y.L. and Y.Z.; writing—review and editing, Y.L.;
supervision, F.Y. All authors have read and agreed to the published
version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The datasets generated and analyzed in this study are available at
PRJNA837352
([143]https://www.ncbi.nlm.nih.gov/bioproject/?term=prjna837352),
accessed on 12 May 2022.
Conflicts of Interest
The authors declare no conflict of interest.
Sample Availability
Not available.
Funding Statement
This research was funded by the Joint Research Project Based on
Cooperative Program for Bachelor of Science in Forestry by Nanjing
Forestry University and the University of British Columbia, A Project
Funded by the Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD) and Undergraduate Research & Practice
Innovation Program of Jiangsu Province (No. 202110298114Y).
Footnotes
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claims in published maps and institutional affiliations.
References