Cloning and expression of floral organ development-related genesin herbaceous peony (Paeonia lactiflora Pall.)

Jintao Ge • Daqiu Zhao • Chenxia Han •

Jing Wang • Zhaojun Hao • Jun Tao

Received: 12 April 2013 / Accepted: 19 June 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Herbaceous peony (Paeonia lactiflora Pall.) is

an important ornamental plant that has different flower

types. However, the molecular mechanism underlying its

floral organ development has not been fully investigated.

This study isolated six floral organ development-related

genes in P. lactiflora, namely, APETALA1 (PlAP1), A-

PETALA2 (PlAP2), APETALA3-1 (PlAP3-1), APETALA3-

2 (PlAP3-2), PISTILLATA (PlPI) and SEPALLATA3

(PlSEP3). The expression patterns of these genes were also

investigated in the three cultivars ‘Hangshao’, ‘Xiangy-

angqihua’ and ‘Dafugui’. Furthermore, gene expression

during floral development was also analyzed in different

organs. The results showed that PlAP1 was mainly

expressed in the sepals, and PlAP2 was mainly expressed

in the carpels and sepals. PlAP3-2 and PlPI had the highest

expression levels in the stamens, followed by the petals.

The expression levels of PlAP3-1 (from highest to lowest)

were in the following order: petals, stamens, carpels and

sepals. PlSEP3 was mainly expressed in sepals and carpels.

With the depth of stamen petaloidy, the expression levels

of PlAP1, PlAP2 and PlSEP3 increased, whereas those of

PlAP3-1, PlAP3-2 and PlPI decreased, which showed that

PlAP1 mainly determined sepals and petals of P. lactiflora.

The PlAP2 not only determined the sepals and petals, and it

participated in carpel formation. PlAP3-1, PlAP3-2 and

PlPI mainly determined stamens and petals. PlSEP3

determined the identities of sepals and petals. This study

would help determine the molecular mechanism underlying

floral organ development in P. lactiflora.

Keywords Paeonia lactiflora � Floral organ

development � Cloning � Gene expression

Introduction

Herbaceous peony (Paeonia lactiflora Pall.), which belongs

to Paeoniaceae family, is a traditional rare flower in China

and is widely cultivated worldwide, with more than

3,900 years of cultivation history. For long-term adaptation

to natural and artificial selections, more than 600 cultivars

have been authenticated at present [1]. P. lactiflora can be

classified according to flower shape: single-petal and double-

petal categories. The latter can be further classified into

lotus-shaped, chrysanthemum-shaped, rose-shaped, anem-

one-shaped, golden circle-shaped, crown-shaped, hydran-

gea-shaped and proliferate flower-shaped [2].

According to previous studies, the double flower was

derived through three pathways. One evolved through

increases in the number of petals or floral whorls, such as

double balsamine (Impatiens balsamina L.). The second

developed from the replacement of tubular flowers with

lingulate flowers in the capitulum or the area of tubular

flowers increased, such as in chrysanthemum (Dendrant-

hema morifolium Tzvel.). The third formed from the

increased area of petals through extreme folding, such as

double corn poppy (Papaver rhoeas L.) [3]. Double her-

baceous peonies were derived from the first pathway,

which relies on stamen petaloidy to increase the petal

Jintao Ge and Daqiu Zhao contributed equally to this work.

J. Ge � D. Zhao � C. Han � J. Wang � Z. Hao � J. Tao (&)

Jiangsu Key Laboratory of Crop Genetics and Physiology,

College of Horticulture and Plant Protection, Yangzhou

University, Yangzhou 225009, People’s Republic of China

e-mail: taojun@yzu.edu.cn

J. Ge

Lianyungang Academy of Agricultural Sciences,

Lianyungang 222006, People’s Republic of China

123

Mol Biol Rep

DOI 10.1007/s11033-014-3532-8

numbers. Lotus-shaped, chrysanthemum-shaped, rose-

shaped, and proliferate flower-shaped flowers exhibit cen-

tripetal petaloidy, whereas anemone-shaped, golden circle-

shaped, crown-shaped, and hydrangea-shaped flower

exhibit centrifugal petaloidy [2].

The formation pattern, development process, and flower

structure have strong hereditary stability. They are the most

important bases for angiosperm phylogeny and develop-

ment research, are crucial characteristics to which classic

taxonomists rely on. Therefore, floral organs are the ideal

model system for studying the correlation between plant

development, gene and evolution [4].

In 1991, Coen et al. [5] proposed the ABC model of floral

organ development by studying flower homologous deformed

mutants in arabidopsis [Arabidopsis thaliana (L.) Heynh.]

and snapdragon (Antirrhinum majus L.). The model sug-

gested that the floral organ of A. thaliana could be divided into

4 whorls, namely, sepals, petals, stamens, and carpels (from

the outside to the inside), which were respectively determined

by A-, B-, and C-class genes. A-class genes determined the

formation of sepals while A-class genes and B-class genes

controlled petal formation. B-class and C-class genes deter-

mined stamen formation, and C-class genes determined car-

pel formation. In addition, A-class genes were antagonistic to

C-class genes. A-class gene mutations induced sepals and

petals to develop into carpels and stamens, respectively,

whereas C-class gene mutations induced stamens and carpels

to develop into petals and sepals, respectively.

Although A-, B-, and C-class genes determined the

formation of floral organs, Bowman et al. found that leaves

did not developed into flowers through their overexpression

in leaves. Accordingly, other genes also determined floral

organ formation [6]. In 1995, Angenent et al. [7] found the

genes FLORAL BINDING PROTEIN7 (FBP7) and FBP11,

which controlled ovule development in petunia (Petunia

hybrida Vilm.). Colombo introduced the ABCD model of

floral organ development. Genes that determined ovule

development were classified as D-class genes, which had a

redundant effect as C-class gene [8]. In 2000, Pelaz et al.

found SEPALLATA1/2/3 (SEP1/2/3) genes from A. thali-

ana. When SEP1/2/3 genes mutated at the same time, each

whorl floral organ developed into sepal-like structures [9,

10]. Thus, the three genes and SEP4, which regulated floral

organ development, were classified as E-class genes, and

the ABCDE model of the floral organ development was

established. To date, the genetic and molecular study of

double flowers focuses on the ABCDE model.

The A-class genes in A. thaliana had already been iso-

lated, including the APETALA1 (AP1) and APETALA2

(AP2) genes [11]. The B-class genes included the APET-

ALA3 (AP3) and PISTILLATA (PI) genes [12], and the

C-class genes only included the AGAMOUS (AG) gene

[13]. The D-class genes included SEEDSTICK (STK) and

SHATTERPROOF1/2 (SHP1/2) and included FBP7 and

FBP11 class genes in hybrids [8]. The E-class genes

included SEP1-SEP4 [9, 10, 14, 15].

As studies continued, the other functions of the afore-

mentioned five categories were also identified, which

affected some physiologic processes. AP1 had important

function in regulating inflorescence meristem transforma-

tion and promoting early blooming [16, 17]. AP2 protein

was widely existed in plants and regulated plant develop-

ment and growth. AP2 family resisted ethylene, abscisic

acid, and pathogen infection [18]. Moreover, PI played an

important role in floral organ development and affected the

whole plant growth as observed in the butterfly orchid PI

transformation into tobacco [19]. To regulate stamen and

carpel development, the C-class gene AG also maintained

the identity of floral meristem with LEAFY (LFY) [20].

Meanwhile, AG suppressed the expression of WUSCHEL

(WUS) gene to terminate the activity of floral meristem by

reducing the number of blooms and removaling of carpels

and stamens in some flowers [21].

The merits and defects of flower type, which are directly

related to the ornamental and commercial value of plant, are

an important quality index for ornamental plants. Thus,

breeding new cultivars with different flower types is a hot-

spot in ornamental plant breeding. P. lactiflora lacks peculiar

and excellent flower cultivars, but it is popular worldwide.

Molecular breeding had become an important method for

cultivating new cultivars of P. lactiflora. This technique

shortens breeding period, expands the plant gene bank, and

induces directional gene modification. However, studies on

P. lactiflora genes related to floral organ development are not

available. Therefore, study on the molecular mechanism of

flower type formation is important will serve as a foundation

for transgenic technology to cultivate new cultivars.

This study adopted Rapid Amplification of cDNA Ends

(RACE) technology to isolate A-class genes PlAP1 and

PlAP2; B-class genes PlAP3-1, PlAP3-2 and PlPI; and

E-class gene PlSEP3 in P. lactiflora, which are related to

flower type formation. The expression pattern study of floral

organ development-related genes in different developmental

stages and different cultivars using real-time quantitative

polymerase chain reaction (Q-PCR) technology was in order

to clarify their roles in the formation of P. lactiflora flower

type, find out the key genes, which could provide a theo-

retical basis for breeding new P. lactiflora cultivars.

Materials and methods

Plant materials

The normal petals of ‘Xiangyangqihua’ (anemone-shaped)

were selected to clone the gene, and the petals of ‘Hangshao’

Mol Biol Rep

123

(single flower), ‘Xiangyangqhua’ (anemone-shaped), and

‘Dafugui’ (proliferate flower-shaped) were chosen for gene

expression (Fig. 2). All the plants were grown in the germ-

plasm repository of Horticulture and Plant Protection Col-

lege, Yangzhou University, Jiangsu Province, People’s

Republic of China (32�300N, 119�250E). Flowers of

‘Hangshao’ during four development stages including

flower-bud stage (S1), initiating bloom stage (S2), bloom

stage (S3) and wither stage (S4) were collected from March

to May 2012. These flowers in S3 were divided into four

parts: sepals, petals, stamens and carpels. The petals of

‘Xiangyangqihua’ and ‘Dafugui’ in S3 were collected, and

petals of ‘Xiangyangqihua’ were divided into three parts:

primary petaloid stamens, moderate petaloid stamens, and

normal petals (Fig. 1). All samples were immediately frozen

with liquid nitrogen and stored at -80 �C until analysis.

RNA extraction and purification

Total RNA was extracted according to a modified CTAB

extraction protocol [22]. Prior to reverse transcription,

RNA samples were treated with DNase using a DNase I kit

(TaKaRa, Japan) according to the manufacturer’s guide-

lines. The samples were then quantified using a spectro-

photometer (Eppendorf, Germany) at 260 nm.

Isolation of genes

30 RACE

First-strand cDNA was synthesized from the total RNA of

the petal tissues (1 lg) using a 30 full RACE Core Set

Ver.2.0 (TaKaRa, Japan) according to the manufacturer’s

instructions. The 30 ends of the cDNA were amplified

through two rounds PCR with gene-specific primers

(Table 1). These primers were designed according to the

floral organ development genes of other plants from the

GenBank database, which included AP1, AP2, AP3, PI, and

SEP3. The first PCR was implemented by denaturing the

cDNA at 94 �C for 3 min, followed by 20 cycles of 30 s at

94 �C, 30 s at 50 �C, 90 s at 72 �C (for AP1), and by a final

extension of 10 min at 72 �C. The nested PCR amplifica-

tion used the first round PCR product as template at the

annealing temperature of 51 �C (for AP1), and 30 cycles

were run under the PCR conditions as the primary PCR.

50 RACE

The first-strand cDNA was synthesized from the total RNA

of petal tissues (1 lg) according to the manufacturer’s

guidelines of the SMARTerTM RACE cDNA Amplification

Fig. 1 Figures of P. lactiflora samples used in this study. The upper

pictures are three P. lactiflora cultivars and the bottom pictures are

three different petals from ‘Xiangyangqihua’. a ‘Hangshao’,

b ‘Xiangyangqihua’, c ‘Dafugui’, d Primary petaloidy stamens,

e Moderate petaloidy stamens, f Normal petals

Mol Biol Rep

123

Kit User Manual (Clontech Laboratories, Inc., TaKaRa).

The gene-specific primers (Table 1) were designed and

synthesized based on the previously mentioned sequenced

30 regions. PCR amplification was performed under the

following conditions: 5 cycles of 94 �C for 30 s and 72 �C

for 90 s, 5 cycles of 94 �C for 30 s, 70 �C for 30 s, and

72 �C for 1 min, and 20 cycles of 94 �C for 30 s, 62 �C for

30 s, and 72 �C for 90 s (for AP1). Table 1 lists the con-

ditions of other gene amplifications.

Purifying, cloning, and sequencing

The PCR products were separated using 1 % agarose gel

electrophoresis, and the incised gels were purified using a

TaKaRa Agarose Gel DNA Purification Kit Ver. 2.0 (Ta-

KaRa, Japan). The extracted products were cloned into

PMD18-T vector (TaKaRa, Japan) and transformed into

competent Escherichia coli DH5a cells (Trans, China). The

recombinant plasmids were identified using the restriction

enzymes BamHI and HindIII (TaKaRa, Japan) and sent to

Shanghai Sangon Biological Engineering Technology &

Services Co., Ltd. (Shanghai, China) for sequencing.

Sequence analysis

Splicing and analysis of PlAP1, PlAP2, PlAP3-1, PlAP3-2,

PlPI, and PlSEP3 sequences were performed using DNA-

MAN 6.0 software. Homology searches were carried out using

the GenBank BLAST (http://blast.ncbi.nlm.nih.gov/Blast/).

Gene expression analysis

Transcription levels of the isolated PlAP1, PlAP2, PlAP3-

1, PlAP3-2, PlPI, and PlSEP3 genes were analyzed using

Q-PCR by a BIO-RAD CFX96TM Real-Time System

(C1000TM Thermal Cycler) (Bio-Rad, USA). The cDNA

was synthesized from 1 lg RNA using PrimeScript RT

reagent Kit with gDNA Eraser (TaKaRa, Japan) and

quantified with a spectrophotometer (Eppendorf, Ger-

many) at 260 nm. P. lactiflora actin (JN105299) was used

as the internal control. All gene-specific primers for

Q-PCR were designed using the Primer 5.0 program

(PREMIER Biosoft International, Canada) (Table 2).

Q-PCR was carried out using the SYBR� Premix Ex

TaqTM (Perfect Real Time) (TaKaRa, Japan) and con-

tained 12.5 ll of 29 SYBR Premix Ex TaqTM, 509 ROX

Reference Dye II 0.5, 2 ll of cDNA solution as a tem-

plate, 2 ll of mix solution of target gene primers, and 8 ll

of ddH2O in a final volume of 25 ll. The amplification

was carried out under the following conditions: 50 �C for

2 min, followed by an initial denaturation step at 95 �C

for 5 min, 40 cycles at 95 �C for 15 s, 51 �C for 15 s, and

72 �C for 40 s. The gene relative expression levels of

target genes were calculated by the 2-44Ct comparative

threshold cycle (Ct) method [23], and the PlAP1 expres-

sion levels in the petals during the budding period

(‘Hangshao’) was used as the control. The Ct values of the

triplicate reactions were obtained using the Bio-Rad CFX

Manager V1.6 .541.1028 software.

Table 1 Primers used for floral

organ development-related

genes isolation in P. lactiflora

Primer Oligonucleotide sequence (50–30) Application Annealing condition

Temperature

(�C)

Time

(s)

AP11 AATACTCCAAACTTAGGGC 1st of 30 RACE 51 90

AP12 GAGGTTGCTTTGATTGTC 2nd of 30 RACE 50 90

AP13 TTTCTCCAGTTGATTCATGCACCAAAGC 50 RACE (Clontech) 62 120

AP21 GGTGGATTTGACACTGCG 1st of 30 RACE 51 120

AP22 CGGGGAGTAGAGGCAGACAT 2nd of 30 RACE 50 120

AP23 AGCTGCATGGTTTCGTTTGCCTCAC 50 RACE (Clontech) 62 120

AP3-11 AGTCCTTCCACTACAACG 1st of 30 RACE 51 60

AP3-12 GAGATTAGGCAGAGGATG 2nd of 30 RACE 50 60

AP3-13 CAGGCGGAAAGCATACAAATTAGTGGC 50 RACE (Clontech) 62 90

AP3-21 AGACCGATCTGTGGAAAT 1st of 30 RACE 51 60

AP3-22 ACCCAGACTGATACCTACA 2nd of 30 RACE 50 60

AP3-33 CATGGTCGCTTGAATGTAGGCGGAAA 50 RACE (Clontech) 62 90

PI1 TGGGAAGAGGTAAGATTG 1st of 30 RACE 51 60

PI2 GCCCTTCTACTACGGTGAT 2nd of 30 RACE 50 60

PI3 TTTCAAGGGTTTCCTCTAGGGCTATGAGC 50 RACE (Clontech) 62 90

SEP31 TTTCCGTCCTTTGCGATG 1st of 30 RACE 51 60

SEP32 TTGGTGAGGACCTTGGAC 2nd of 30 RACE 50 60

SEP33 CTTGGTCCTGCCGCTGCTACTCCTAT 50 RACE (Clontech) 62 90

Mol Biol Rep

123

Statistical analysis

All data were the average of at least three replicates with

standard deviations.

Results

Isolation and sequence analysis of floral organ

development-related genes

We successfully isolated the full-length cDNA of six floral

organ development-related genes that encoded PlAP1,

PlAP2, PlAP3-1, PlAP3-2, PlPI, and PlSEP3 from P.

lactiflora using RACE technology (Tables 3, 4). The

1,106 bp PlAP1 cDNA contained a 729 bp open reading

frame (ORF) that encoded a 242-amino acid protein.

BLAST analysis showed that this protein shared 52–89 %

identity and 76–93 % similarity with AP1 from Paeonia

suffruticosa, Vitis vinifera, Heuchera americana, Coryl-

opsis sinensis, and Mangifera indica. Similarly, the

1,935 bp PlAP2 cDNA contained a 1,575 bp ORF that

encoded a 524-amino acid protein. BLAST analysis

showed that this protein shared 42–75 % identity and

71–85 % similarity with AP2 from V. vinifera, Camellia

sinensis, Populus trichocarpa, Ricinus communis, and

Glycine max. The 895 bp PlAP3-1 contained a 663 bp

ORF that encoded a 220-amino acid protein. BLAST

analysis showed that this protein shared 61–82 % identity

and 75–95 % similarity with AP3 from P. suffruticosa, V.

vinifera, Corylopsis pauciflora, Actinidia chinensis, and

Davidia involucrata. The 844 bp PlAP3-2 contained a

663 bp ORF that encoded a 220-amino acid protein.

BLAST analysis showed that this protein shared 64–81 %

identity and 75–97 % similarity with AP3 from P. suffr-

uticosa, C. pauciflora, V. vinifera, A. chinensis, and Sar-

racenia. The 890 bp PlPI contained a 627 bp ORF that

encoded a 208-amino acid protein. BLAST analysis

showed that this protein shared 64–74 % identity and

76–80 % similarity with PI from V. vinifera, A. chinensis,

Camellia japonica, Prunus avium, and Nyssa sylvatica.

The 1,165 bp PlSEP3 contained a 729 bp ORF that enco-

ded a 242-amino acid protein. BLAST analysis showed that

this protein shared 60–62 % identity and 80–81 % simi-

larity with PI from P. avium, Populus tremuloides, V.

vinifera, Prunus persica, and Populus tomentosa.

These cloned genes had been deposited in the GenBank

database under accession numbers KC354376, KC455454,

KC354377, KC354378, KC354379, and KC354380,

respectively.

Expression analysis of floral organ development genes

To identify the regulatory mechanism of floral organ

development genes in different floral organs of ‘Hang-

shao’, six isolated floral organ development-related genes

were used to detect their expression levels in different

floral organs according to Q-PCR technology.

Figure 2 shows that the A-class gene PlAP1 had the

highest expression levels in sepals, followed by petals, and

had the lowest expression level in carpels and stamens. The

PlAP1 expression levels in the sepals were nearly 100

times that in stamens. The PlAP2 gene had the highest

expression levels in the carpels, followed by the sepals, and

Table 2 Gene-specific primers

sequence for detection by

Q-PCR

Gene Forward primer (50–30) Reverse primer (50–30)

Actin ACTGCTGAACGGGAAATT ATGGCTGGAACAGGACTT

AP1 AAACCAAAGGCACTTTATG CATTGCCGTTTCCTTCTT

AP2 GCAAGTCGGCGGAGGTTT TCCCATCGGCCAGTTCTA

AP3-1 TCCCATTGGAGGTGATTT TTGCGACGCTTTGAGTAA

AP3-2 CCATAATACGCCAACAAA CTTCAAAGCCTAGCAGGA

PI CAGCCTATCCAGCCAAAT CAAGTTCACCACGCCTTA

SEP3 TTGGTGAGGACCTTGGAC CTTTGTTTGAGGCTTTGATT

Table 3 Sequence analysis of

floral organ development-

related genes in P. lactiflora

Gene Full length

(bp)

ORF

(bp)

30-UTR

(bp)

50-UTR

(bp)

Poly

A (bp)

Amino

acid

Molecular

weight (kDa)

PlAP1 1,106 729 207 120 12 242 28.07

PlAP2 1,935 1,575 137 222 12 524 86.81

PlAP3-1 895 663 169 62 22 220 25.52

PlAP3-2 844 663 110 71 11 220 25.36

PlPI 890 627 240 22 12 208 24.30

PlSEP3 1,165 729 265 180 12 242 27.77

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had lowest expression levels in the petals and stamens. The

expression level of PlAP2 in carpels was 100 times higher

than that in stamens. The expression of two B-class genes

PlAP3-2 and PlPI were in accordance with the trend. They

had the highest expression levels in the stamens, followed

by the petals, and had lowest expression level in carpels

and sepals. The expression level of PlAP3-2 in four-wheel

floral organs were 100 times higher than that of PlAP3-1.

Meanwhile, the expression levels of the PlAP3-1 gene from

highest to lowest were as follows: petals, stamens, carpels,

and sepals. The difference in expression levels was small.

The expression levels of the PlSEP3 gene from highest to

lowest was as follows: sepals, carpels, petals, and stamens.

The expression levels in the sepals were nearly 100 times

that in the stamens. In general, the expression levels of the

PlAP3-2, PlPI, and PlSEP3 genes were higher than A-class

gene.

This paper investigated the expression level of six genes

of herbaceous peony floral organ development in the petals

of ‘Hangshao’ at four developmental stages (Fig. 3). The

expression level of each gene was stable, and those of the

B-class gene PlPI was higher than the other genes. In

A-class genes, the expression level of PlAP1 gene was

highest in S1 and lowest in S2, but PlAP2 gene was basi-

cally decreased. Among the B-class genes, the gene

expression level of PlAP3-1 was highest in S1 and lowest

in S2, which is consistent with that of PlAP1. The

expression levels of the former were about 1.6 times the

height of the latter. The PlAP3-2 gene expression levels

were also highest in S1 and lowest in S2, PlAP3-2 gene

presented a decreasing trend in S3 and S4. PlPI gene

expression levels were different from those of the first four

genes, with the highest expression observed in S2, which

then declined, and the lowest was during the budding stage.

The expression of the E-class gene PlSEP3 showed an

increasing trend during flower deveolpment.

Figure 4 showed the expression analysis of the six floral

organ development genes in the petals of three P. lactiflora

cultivars ‘Hangshao’, ‘Xiangyangqihua’ and ‘Dafugui’. In

general, the expression levels of the B-class genes were

significantly higher than those of the A-class genes. The

expression levels of the E-class gene was between the two

previously mentioned peaks. The expression levels of the

B-class genes PlAP3-2 and PlPI in ‘Xiangyangqihua’ were

lower than those in ‘Hangshao’ and ‘Dafugui’. The

expression levels of PlAP3-2 in ‘Hangshao’ and ‘Dafugui’

were five times higher than that in ‘Xiangyangqihua’. The

expression levels of PlPI in ‘Hangshao’ and ‘Dafugui’

were two and three times higher than that in ‘Xiangy-

angqihua’, respectively. Moreover, the expression levels of

the A-class gene PlAP1 in ‘Hangshao’ was three times

higher than that in ‘Dafugui’. The three other genes did not

exhibit difference, which were more than doubled.

No obvious changing trends were noted among the

different flower organs, developmental stages and cultivars.

Determining the changes in each gene during floral organ

development was difficult because the control groups have

too many variables. Thus, we selected three different petals

Table 4 Comparsion of deduced floral organ development-related

genes protein in P. lactiflora with other plants

Gene Species GenBank

accession no.

Identity

(%)

Similarity

(%)

PlAP1 Paeonia

suffruticosa

HM143943.1 83 93

Vitis vinifera XM_002263134.1 58 82

Heuchera

americana

AY306148.1 55 79

Corylopsis

sinensis

AY306146.1 52 79

Mangifera

indica

GQ152892.1 59 76

PlAP2 V.vinifera XM_002283009.2 75 85

Camellia

sinensis

JQ398741.1 75 71

Populus

trichocarpa

XM_002310679.1 56 73

Ricinus

communis

XM_002534353.1 48 76

Glycine max XM_003524595.1 42 78

PlAP3-

1

P. suffruticosa DQ479364.1 82 95

V.vinifera DQ979341.1 69 77

Corylopsis

pauciflora

DQ479354.1 61 79

Actinidia

chinensis

HQ113358.1 69 75

Davidia

involucrata

GQ141153.1 65 76

PlAP3-

2

P. suffruticosa DQ479363.1 81 97

C. pauciflora DQ479354.1 64 79

V.vinifera DQ979341.1 73 76

A. chinensis HQ113358.1 72 75

Sarracenia GQ141165.1 67 75

PlPI V.vinifera DQ059750.1 71 80

A. chinensis HQ113360.1 70 77

Camellia

japonica

HQ141341.1 74 76

Prunus avium HQ229606.1 71 76

Nyssa sylvatica GQ141109.1 64 77

PlSEP3 P. avium HQ229605.1 62 81

Populus

tremuloides

AY235222.1 60 80

V.vinifera XM_002275669.2 61 81

Prunus persica EF440351.1 62 81

Populus

tomentosaDQ445094.1 60 80

Mol Biol Rep

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from ‘Xiangyangqihua’, which only exhibited different

degrees of petaloidy. Figure 5 showed that the expression

levels of B-class genes were higher than those of the

A-class genes. With the depth of stamen petaloidy, the

expression levels of the A-class gene PlAP1 and the

E-class gene PlSEP3 gradually increased, but the expres-

sion levels of the B-class genes PlAP3-2 and PlPI gradu-

ally decreased. The expression levels of PlAP3-2, PlPI, and

PlSEP3 were significantly higher, with the highest

expression five times higher than the lowest.

Discussion

Flowering is an important period of plant growth and

development. It is a complex physiologic and biochemical

process regulated by many genes. This study used RNA

extracted from P. lactiflora petals as a template to isolate

the genes involved in floral organ development according

to RACE technology. Six floral organ development-related

genes were obtained. PlAP1 and PlAP2 are A-class genes,

PlPI is B-class gene and PlSEP3 is an E-class gene.

To date, the ABCDE model is widely used for

explaining the molecular developmental genetic mecha-

nism of floral organ formation. Some researchers reported

that the ABCDE model was found in model species of

eudicot plant; the conservation of the ABCDE model in

different plant populations differed significantly [24].

Kyozuka et al. [25] found that many genes in Oryza sativa

were homologous with to Arabidopsis and Snapdragon

genes, but they had different functions. Therefore, whether

the ABCDE model can be applied to P. lactiflora needs to

be studied.

Previous studies had shown the AP1 gene expression in

the four-wheel floral organs of Prunus lannesiana, Mag-

nolia grandiflora, P. persica, P. suffruticosa [26–29]. In

P. lannesiana, PlAP1 gene expression exhibited no obvious

differences among four-wheel floral organs [26]. In

PlA

P1

rel

ativ

e e

xpre

ssio

n le

vel

0

1

2

3

4

5

6

PlA

P2

rel

ativ

e e

xpre

ssio

n le

vel

0.0

.5

1.0

1.5

2.0

2.5

PlA

P3-

1 r

elat

ive

exp

ress

ion

leve

l

0.0

.2

.4

.6

.8

1.0

PlA

P3-

2 r

elat

ive

exp

ress

ion

leve

l

0

2080

100

120P

lPI

rel

ativ

e e

xpre

ssio

n le

vel

0

50150

200

Different floral organs

sepal petal stamen carpel sepal petal stamen carpel

sepal petal stamen carpel sepal petal stamen carpel

sepal petal stamen carpel sepal petal stamen carpel

PlS

EP

3 r

elat

ive

exp

ress

ion

leve

l

0

50150

200

250

Fig. 2 Expression analysis of

floral organ development-

related genes in different organs

of P. lactiflora (‘Hangshao’)

Mol Biol Rep

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M. grandiflora, the MgAP1 gene exhibited the highest

expression levels in the carpels, and a small amount was

observed in the stamens and the inner perianth [27]. In P.

suffruticosa, the PsAP1 gene exhibited the highest

expression levels in the petals, followed by the carpels and

the sepals [29]. In this study, the PlAP1 gene of herbaceous

peony was expressed in all four whorl flower organs. The

PlAP1 gene had the highest expression levels in the sepals,

followed by petals. This result was consistent with previous

results [30], which showed that PlAP1 gene was mainly

determined in sepals and petals.

The expression pattern of AP2 had been studied in Malus

domestica, Crocus sativus, P. suffruticosa and Nymphaea

alba [29, 31–33]. In N. alba, the NaAP2 gene had the highest

expression level in the petals, followed by the sepals, and

small amounts were found in the stamens and carpels [33]. In

C. sativus, the CsAP2 gene had the highest expression levels

in the carpels, and small amounts were found in the perianths

PlA

P1

rel

ativ

e e

xpre

ssio

n le

vel

0.0

.2

.4

.6

.8

1.0

1.2

1.4

PlA

P2

rel

ativ

e e

xpre

ssio

n le

vel

0.0

.1

.2

.3

.4

PlA

P3-

1 re

lativ

e e

xpre

ssio

n le

vel

0.0

.2

.4

.6

.8

1.0

1.2

1.4

1.6

PlA

P3-

2 r

elat

ive

exp

ress

ion

leve

l

0

2

4

6

8

10

12

14P

lPI

rela

tive

exp

ress

ion

leve

l

0

5

10

15

20

25

30

35

Different developmental stages

PlS

EP

3 r

elat

ive

exp

ress

ion

leve

l

0

1

2

3

4

5

S1 S2 S3 S4 S1 S2 S3 S4

S1 S2 S3 S4 S1 S2 S3 S4

S1 S2 S3 S4S1 S2 S3 S4

Fig. 3 Expression analysis of

floral organ development-

related genes in P. lactiflora

(‘Hangshao’) petals. S1 flower-

bud stage, S2 initiating bloom

stage, S3 bloom stage, S4 wither

stage

PlAP1 PlAP2 PlAP3-1 PlAP3-2 PlPI PlSEP3

Rel

ativ

e ex

pres

sion

leve

l

0

5

10

15

20

25

HangshaoXiangyangqihuaDafugui

Fig. 4 Expression analysis of floral organ development-related genes

in the petals (all petals of one flower as experimental materials) of the

three P. lactiflora cultivars (‘Hangshao’, ‘Xiangyangqihua’ and

‘Dafugui’)

Mol Biol Rep

123

and stamens [32]. In P. suffruticosa, PsAP2 gene had the

highest expression levels in the carpels, followed by the

sepals [29]. The function of the AP2 gene in the carpels had

been extensively studied in A. thaliana. The AP2 gene has an

important function in the development of female gameto-

phytes and ovaries in Arabidopsis [34, 35]. Juan et al. found

that the AP2 gene prevented replum and valve margin

overgrowth, has been incorporated into the current genetic

network as a gene that controls fruit development in A.

thaliana [36]. In this study, the PlAP2 gene in P. lactiflora

was expressed in all four-wheel flower organs. The PlAP2

gene had the highest expression levels in the carpels, fol-

lowed by the sepals, and small amounts were found in the

stamens and petals. Accordingly, the PlAP2 gene mainly

determined carpels and sepals.

The AP3 gene family contains a large number of genes.

Zhou et al. [37] separated three AP3 homologous genes,

namely, BnAP3-2, BnAP3-3, and BnAP3-4 from Brassica

napus. Two AP3 homologous genes were isolated from

Cyclamen persicum and Gerbera jamesonii Bolus,

respectively [38, 39]. The expression levels of these AP3

homologous genes were markedly different. For instance, in

B. napus, three BnAP3 genes were expressed in the petals and

stamens, but their expression levels were significantly dif-

ferent [37]. In C. persicum, CpAP3b expression in the four-

wheel flowers organs was significant, and CpAP3a was

expressed at high levels in the petals and stamens and at a low

level in carpels [38]. This study isolated the two AP3

homologous genes PlAP3-1 and PlAP3-2 from P. lactiflora

but their expression levels greatly differed. Figure 3 showed

that the expression levels of PlAP3-2 were nearly 100 times

higher than that of PlAP3-1; thus, PlAP3-2 played a more

important role than PlAP3-1. In the four-wheel flowers

organs of P. lactiflora, both PlAP3-1 and PlAP3-2 were

highly expressed in the stamens, followed by the petals. This

result was in line with those of previous studies. The

expression of PlAP3-1 and PlAP3-2 decreased during her-

baceous peony stamen petaloidy, which indicated that the

AP3 gene had a dose-dependent effect on stamens and petals.

This result was the same as that reported by Zhang [40].

PlA

P1

rel

ativ

e e

xpre

ssio

n le

vel

0.0

.1

.2

.3

.4

.5

.6

.7

PlA

P2

rel

ativ

e e

xpre

ssio

n le

vel

0.0

.1

.2

.3

.4

.5

.6

PlA

P3-

1 r

elat

ive

exp

ress

ion

leve

l

0.0

.5

1.0

1.5

2.0

2.5

PlA

P3-

2 r

elat

ive

exp

ress

ion

leve

l

0

2

4

6

8

10

12

14

16P

lPI

rel

ativ

e e

xpre

ssio

n le

vel

0

10

20

30

40

50

60

Different petalled degree

D1 D2 D3 D1 D2 D3

D1 D2 D3 D1 D2 D3

D1 D2 D3 D1 D2 D3

PlS

EP

3 r

elat

ive

exp

ress

ion

leve

l

0

1

2

3

4

5

6

7

Fig. 5 Expression analysis of

floral organ development-

related genes in three different

petals of ‘Xiangyangqihua’. D1

primary petaloid stamens, D2

moderate petaloid stamens, D3

normal petals

Mol Biol Rep

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PI gene expression is stable in most plants. The PI gene

was only notably expressed in the petals and stamens of V.

vinifera, C. persicum, P. suffruticosa, G. jamesonii Bolus,

and so on [29, 38, 39, 41]. The results of this study showed

that P. lactiflora PI homologous gene PlPI was highly

expressed in the stamens and petals, which was consistent

with previous studies. The expression levels of PlPI in the

stamens were 11.3 times than that in the petals, and the

expression of PlAP3 and of PlPI gradually decreased

during stamen petaloidy. This finding proved that the

B-class gene expression in stamens was higher than in the

petals, and the reduction in B-class gene expression results

in stamen petaloidy.

Previous study had demonstrated four SEP3 genes,

namely, CsSEP3a, CsSEP3b, CsSEP3c, and CsSEP3c

expressed in the four-wheel floral organs of C. sativus L.

[42]. In P. persica, the PpSEP3 expression was high in the

calyx, the stamens, and the carpels, but low in the petals

[43]. In Akebia trifoliata, Shan [44] found the expression of

the SEP3 homologous gene AtSEP3 in the stamens, car-

pels, and developing blades. In Taihangia rupestris, Wang

[45] isolated and cloned the SEP3 homologous genes

TrSEP3 and TrSEP3 and found that they were only

expressed in the petals and stamens of mature flowers. In P.

lactiflora, the expression of the PlSEP3 gene in the calyx

was higher than in the petals and stamens. This finding

indicated that the PlSEP3 gene was mainly involved in

calyx formation by forming tetramers with A-class genes,

and the E-class genes SEP1–SEP4 were speculated to have

different functions. Uimari et al. [46] reached a similar

conclusion. Gerbera hybrida has two E-class genes,

namely, GRCD1 and GRCD2. GRCD1 only participated in

the formation of stamens, whereas GRCD2 was involved in

the formation of the inflorescence meristem, the floral

meristem, and the carpels. This result indicated that E-class

genes had different degrees of subfunctionalization and

neofunctionalization.

The change in gene expression in the three different

types of petals showed that the expression of AP1 and SEP3

gradually increased, whereas that of AP3-2 and PI is

gradually decreased with increasing stamen petaloidy. The

AP1, AP3-2, PI, and SEP3 genes are involved in controlling

stamen petaloidy. Whether the increase in AP1 expression

in the stamens leads to stamen petaloidy remains unclear.

Conclusion

In this study, six floral organ development-related genes in

P. lactiflora were cloned using RACE technology. PlAP1

and PlAP2 were classified as A-class genes; PlAP3-1,

PlAP3-2 and PlPI were classified as B-class genes; and

PlSEP3 was classified as an E-class gene. These six genes

had different functions during flower organ formation.

PlAP1 was mainly detected in sepals and petals of P.

lactiflora. PlAP2 was found in sepals and petals, and par-

ticipated in carpel formation. PlAP3-1, PlAP3-2 and PlPI

were mainly detected in the stamens and petals; and

PlSEP3 was detected in the sepals and petals.

Acknowledgments This study was financially supported by Agri-

cultural Science & Technology Independent Innovation Fund of Ji-

angsu Province (CX(12)2019) and a project funded by the Priority

Academic Program Development of Jiangsu Higher Education

Institutions.

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