horticulturae

Article
Genome-Wide Identification and Expression Analysis of
CAMTA Gene Family Implies PbrCAMTA2 Involved in Fruit
Softening in Pear
Jinshan Yu 1,† , Bobo Song 1,† , Kaidi Gu 2 , Beibei Cao 1 , Kejiao Zhao 1 , Jun Wu 1, * and Jiaming Li 1, *

1 State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization,
Nanjing Agricultural University, Nanjing 210095, China
2 National Key Laboratory of Crop Biology, College of Horticulture Science and Engineering,
Shandong Agricultural University, Tai’an 271018, China
* Correspondence: [email protected] (J.W.); [email protected] (J.L.); Tel.: +86-25-84396485 (J.W. & J.L.)
† These authors contributed equally to this work.

Abstract: CAMTA are calcium-modulating binding transcription factors that contribute to plant
development. We identified 46 CAMTA genes from eight Rosaceae species and divided them into
five subgroups based on a phylogenetic tree. Our analysis indicated that CAMTA is a highly
conserved family among Rosaceae species, with a conserved DNA-binding domain (CG-1) and a
conserved transcription factor immunoglobulin domain (TIG). Following a recent whole-genome
duplication event, the genomes of Chinese white pear, European pear, and apple experienced
significant expansion, resulting in the number of CAMTA genes being twice that of the other species.
Cis-element identification showed that the distribution of the zein metabolism regulation-responsive
element was different in the promoters of Chinese white pear (55.56%) and European pear (11.11%)
CAMTA gene families. The gene expression results showed that PbrCAMTA1, 2, 6, 7 was highly
expressed in pear fruit. Among them, PbrCAMTA2 may have a key influence on fruit softening, as
observed in transient transformation experiments. In conclusion, our results provide crucial insights
into the evolution of the CAMTA gene family in pear and other Rosaceae species and identify a
candidate PbrCAMTA gene, which is involved in the dynamic development of pear fruits.
Citation: Yu, J.; Song, B.; Gu, K.;
Cao, B.; Zhao, K.; Wu, J.; Li, J.
Keywords: pear; CAMTA; synteny analysis; qRT-PCR; fruit softening
Genome-Wide Identification and
Expression Analysis of CAMTA Gene
Family Implies PbrCAMTA2 Involved
in Fruit Softening in Pear.
1. Introduction
Horticulturae 2023, 9, 467. https://
doi.org/10.3390/horticulturae9040467 As the third most significant perennial fruit species, pears provide nutrients, antiox-
idants, and dietary fiber and are commonly used in traditional Chinese medicine. Pears
Academic Editor: Sherif M. Sherif
can be divided into two groups: European pears and Asian pears [1]. After harvesting,
Received: 4 March 2023 most European pear species’ flesh becomes soft quickly, and storability is poor. Some
Revised: 3 April 2023 early ripening pear species have even shorter post-harvest storage periods. We know
Accepted: 5 April 2023 fruit quality and post-harvest storage capabilities are significant factors in the commercial
Published: 7 April 2023 quality assessment of fruit products, while fruit maturity determines the quality of fruit
post-harvest and directly affects the current market and consumer choices [2,3]. Fruit soft-
ening manifests as a loss of fruit firmness, which is thought to be due to ethylene-mediated
enzymatic modification of the cell wall, such as β-galactosidase (TBG), xyloglucan endo-
Copyright: © 2023 by the authors.
transglucosylase (XET), and Polygalacturonase (PG) [4,5]. Pectin galactose is hydrolyzed
Licensee MDPI, Basel, Switzerland.
into glucose and galactose by β-galactosidase, and previous studies have reported that
This article is an open access article
when loss of firmness is detected during persimmon fruits ripening, β-galactosidase ac-
distributed under the terms and
tivity increases almost 3.7-fold [6]. XET is associated with the swelling and loosening
conditions of the Creative Commons
of the cell wall and shows an increase in activity during ethylene-induced softening in
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
kiwifruit [4]. 1-Aminocyclopropane-1-Carboxylate Oxidase (ACO) participates in ethylene
4.0/).
de novo synthesis, and ACO1 is reported to have low expression levels in green tomatoes,

Horticulturae 2023, 9, 467. https://doi.org/10.3390/horticulturae9040467 https://www.mdpi.com/journal/horticulturae

Horticulturae 2023, 9, 467 2 of 19

with its level increasing rapidly when the fruit begins to ripen [7]. Some transcription
factors are also known to affect fruit ripening, such as calmodulin-binding transcription
activator (CAMTA), which is extremely responsive to ethylene and regulates tomato fruit
ripening [8].
The CAMTA family is a highly conserved gene family in plants. Since it was first
discovered in tobacco [9], the CAMTA gene family has been identified in many other plants,
such as Arabidopsis thaliana [10], Solanum lycopersicum [8], Vitis vinifera [11], Zea mays [12]
Fragaria ananassa [13], Populus trichocarpa [14], Gossypium [15], Citrus sinensis [16], Musa
acuminata [17], and Linum usitatissimum [18]. CAMTA plays many significant roles in plants,
and it has been reported to have a wide range of functions. CAMTA has been discovered
in ethylene-induced conditions [19]: NtER1/CAMTA increases with ethylene induction in
tobacco, and the content of CAMTA is significantly higher in senescent leaves than in young
leaves [9]. AtSR1/AtCAMTA3 directly regulates NDR1 and EIN3 and is involved in the plant
ethylene-related aging process [20]. After treating tomato with exogenous ethylene, there is
a significant increase in the expression of seven SlCAMTA/SR genes in tomatoes, suggesting
that CAMTA might act as an ethylene responder to regulate fruit ripening [8]. In addition,
the expressions of PG, PE1, CEL2, and other fruit wall metabolic genes are promoted in
SlSR4 mutants, suggesting that CAMTAs are involved in the tomato fruit softening process.
CAMTA genes respond to different biotic and abiotic stresses. AtCAMTA3 is involved in
the regulation of the cold response of Arabidopsis by positively regulating the expression
of CBF2 and improving cold resistance [21]. CAMTA genes mediate SA biosynthesis in
Arabidopsis thaliana [22]. These studies have proved that the CAMTA family has major
effects on plant growth, fruit maturation, and stress regulation.
Despite systematic functional research related to CAMTA genes being extensive in a
number of different plants, the PbrCAMTA gene family in pear has not yet been researched.
Thanks to the publication of the genome sequence of P. bretschneideri [23], it is now pos-
sible to carry out the relevant research. Here, we used eight Rosaceae genomes, Pyrus
bretschneideri (Chinese white pear), Pyrus communis (European pear), Fragaria vesca (straw-
berry), Prunus mume (Japanese apricot), Prunus persica (peach), Rubus occidentalis (black
raspberry), Malus domestica (apple), Prunus avium (sweet cherry), and two identified model
plants (Arabidopsis thaliana [10] and Tomato [8])to cluster and analyze the CAMTA protein
family. We performed analysis on genome-wide identification, phylogenetic relationship,
chromosome distribution, genomic structure, expression patterns, and function verification
of PbrCAMTA genes. Our findings have the potential to lead to a better understanding of
the roles and structures of CAMTA genes in Rosaceae species, especially pear, and can be
useful for screening candidate genes that may be associated with fruit softening, this way
laying the foundation for function identification.

2. Materials and Methods

2.1. Whole-Genome Identification of CAMTA Genes
The genome data of eight Rosaceae species were downloaded via the GDR database
(https://www.rosaceae.org/, accessed on 5 December 2021). We then used two differ-
ent methods to identify the genes of the CAMTA family. Firstly, the Pfam file of the
CAMTA domain was used to identify candidate CAMTA genes in eight Rosaceae species
(Chinese white pear, European pear, Strawberry, Japanese apricot, peach, black rasp-
berry, apple, and sweet cherry) using HMMSearch software. The protein sequences of the
CAMTA family of Arabidopsis and tomato were subsequently downloaded [8,10]. Using the
CAMTA protein sequences in Arabidopsis and tomato as a query, we identified candidate
CAMTA genes in the genomes of eight Rosaceae species using the blastp software. We
used the overlapping results of both methods for further analysis. We employed the CDD
tool (https://www.ncbi.nlm.nih.gov/cdd/, accessed on 10 December 2021), SMART tool
(http://smart.emblheidelberg.de/, accessed on 10 December 2021), and InterProScan tool
(http://www.ebi.ac.uk/Tools/pfa/iprscan/, accessed on 10 December 2021) to verify the
completeness of the CAMTA domain and to perform a functional analysis of the correspond-
Horticulturae 2023, 9, 467 3 of 19

ing protein sequence. In addition, we used the Expasy tool (https://www.expasy.org/,

accessed on 5 December 2021) to predict protein molecular weights (Mw) and isoelectric
points (PI) for the eight species considered.

2.2. Multiple Sequence Alignment and Phylogenetic Tree Analysis

We utilized ClustalW with the default parameters to align multiple protein sequences
in ten species (eight Rosaceae species, Arabidopsis, and tomato) and then used the Genedoc
software to visualize the results of multiple sequence alignment. Using the multi-sequence
alignment result, the neighbor-joining method (NJ) was used, 1000 bootstraps were set,
and an unrooted phylogenetic tree was constructed with MEGA-X software.

2.3. Gene Structure Analysis and Conserved Motifs Analysis

We obtained CAMTA gene structure information and chromosome position informa-
tion of the CAMTA genes of eight Rosaceae species from genome databases using our
in-house Python scripts. The gene structure of the CAMTA genes in Rosaceae species was
visualized with the TBtools tool [24]. The MEME tool (Multiple Em for Motif Elicitation)
(http://meme-suite.org/tools/meme/, accessed on 30 December 2021) was used to iden-
tify conserved motifs, and the parameters were set as follows: repetitions, any number; the
number of different motifs, 20; minimum motif width, 30; and maximum motif width, 70.
We obtained merged visualizations through the iTOL tool.

2.4. Chromosomal Location and Synteny Analysis

Chromosome location information of CAMTA genes was visualized with TBtools [24].
Homologous gene pairs were identified using the MCScanX software, and the circle picture
was generated by the Circos software (version 0.69.2).

2.5. Cis-Regulatory Elements Analysis of Putative Promoters

All promoter sequences of CAMTA genes were extracted using the “getfasta” function
in Bedtools to extract 2000 bp sequences upstream of the transcriptional initiation sites
(putative promoter regions). We then used PlantCARE (http://bioinformatics.psb.ugent.
be/webtools/plantcare/html/, accessed on 18 January 2022) to predict cis-regulatory
elements of the PbrCAMTA gene promoter regions.

2.6. Transcriptome Expression Pattern Analysis

We used the transcriptome data from six fruit developmental periods of ‘Dangshansuli’
cultivar (15 DAFB (days after full bloom), 36 DAFB, 80 DAFB, 110 DAFB, 145 DAFB, and 167
DAFB, which correspond, respectively to DS1, DS2, DS3, DS4, DS5, DS6 in ‘Dangshansuli’),
the transcriptome data from seven fruit developmental periods of other five pear cultivar
(‘Hosui’, ‘Kuerlexiangli’, ‘Nanguoli’, ‘Starkrimson’ and ‘Yali’), and the transcriptome data
from six tissues (leaf, fruit, petal, sepal, ovary and stem) of ‘Dangshansuli’ cultivar. These
samples were obtained from previous studies of our research group [25–27]. Based on
the RPKM value (Reads Per Kilobase Per Million Mapped Reads) of the PbrCAMTA gene,
log2 (RPKM+0.01) was calculated, and the heatmap was drawn using the “pheatmap”
function in R.

2.7. Quantitative Real-Time PCR Analysis (qRT-PCR)

We collected samples from six tissues (leaf, fruit, petal, sepal, ovary, and stem) and fruit
tissues at six developmental periods (15 DAFB, 36 DAFB, 80 DAFB, 110 DAFB, 145 DAFB
and 167 DAFB) of ‘Dangshansuli’ in order to perform qRT-PCR analysis to validate the
results of transcriptome data. The RNA was extracted and reverse transcribed to synthesize
cDNA (TransGen Biotech Co. Ltd., Beijing, China), primers were designed using the NCBI
website (https://www.ncbi.nlm.nih.gov/tools/primerblast, accessed on 8 December 2021),
and the corresponding candidate gene sequences were amplified. qRT-PCR analysis was
performed using LightCycler 480 SYBR GREEN I Master (Roche, Beijing, China). The
Horticulturae 2023, 9, 467 4 of 19

analysis was conducted using three biological and three technical repeats. The 2−∆∆Ct
method was used to calculate the relative gene expression levels in different samples, and
all of the data were normalized based on stem or 15 DAFB samples.

2.8. Transient Transformation of Pear Fruits

To transiently overexpress PbrCAMTAs, we constructed the p1300-35S:PbrCAMTA2-
GFP vector and transferred it into Agrobacterium tumefaciens strain GV3101 using the
freeze–thaw method. We centrifuged and collected the cells and then re-suspended them
in an infiltration buffer mixed with 10 mM MgCl2 , 10 mM MES (pH 5.7), and 200 µM
acetosyringone (AS) to OD600 0.8–1.0 and induced at 20–25 ◦ C for 4 h. The cells were
agro-infiltrated into ‘Zaosu’ pear fruits at 115 DAFB using 1 mL needleless syringes. We
identified six injection locations along the equatorial line of each pear fruit so that the same
fruit could be injected three times with PbrCAMTA2 and three times with an empty vector.
Five fruits were injected for each experiment. The injected pear fruits were placed overnight
in the dark at 25 ◦ C and incubated at 25 ◦ C constant temperature for five days before being
examined. Three biological replicates were taken. After measuring the firmness, the fruit
tissue was collected 1–2 cm deep from the injection site and stored at −80 ◦ C.

2.9. Firmness Measurement

A probe compressed the pear fruit tissues near the injection holes at an equatorial
position at 4 mm and 1.5 mm/s speed to obtain the mean peak force using the Brookfield
CT3 Texture Analyzer. The measurement was taken on three experiment-injected holes
and three empty vector-injected holes in each fruit. Five pear fruits were used as biological
replicates in the experiment, and the unit (kg/cm2 ) was used.

3. Results
3.1. Genome-Wide Identification of the CAMTA Gene Family in Chinese White Pear and Other
Rosaceae Species
We identified 46 CAMTA genes from eight Rosaceae species (nine in European pear,
nine in Chinese white pear, eight in apple, five in peach, four in sweet cherry, four in
Japanese apricot, four in black raspberry, and three in strawberry) (Figure 1). Chinese
white pear, European pear, and apple had double the number of CAMTA genes than the
other Rosaceae species. In order to distinguish members of the CAMTA gene family, we
renamed all CAMTA genes and predicted the molecular weight (Mw) and isoelectric point
(pI) of the CAMTA protein sequences with the ExPASy Proteomics Server (Supplementary
File S6: Table S2). The length of the CAMTA-encoded protein sequences ranged from 854
(PcpCAMTA2) to 2065 (FvH4CAMTA1) amino acids, the protein mass ranged from 96 kD
(PcpCAMTA2) to 233 kD (FvH4CAMTA1), and protein pIs ranged from 5.26 (PavCAMTA3)
to 8.16 (FvH4CAMTA3). In Chinese white pear, the length of the CAMTA-encoded protein
sequences ranged from 865 (PbrCAMTA9) to 1152 (PbrCAMTA6) amino acids, the protein
mass ranged from 97 kD (PbrCAMTA9) to 128 kD (PbrCAMTA6), and protein pIs ranged
from 5.38 (PbrCAMTA7) to 7.13 (PbrCAMTA3).
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Horticulturae 2023, 9, 467 5 of 18 5 of 19

Figure 1. Phylogenetic tree and genome information of CAMTA genes in eight Rosaceae species.
Figure 1. Phylogenetic tree and genome information of CAMTA genes in eight Rosaceae species. The
The pentagram indicates the occurrence
pentagram indicates the of whole-genome
occurrence duplication
of whole-genome eventsevents
duplication (WGD).
(WGD).

3.2. Phylogenetic
3.2. Phylogenetic Analysis of CAMTA Analysis of CAMTA
Protein Protein
Family Family
in Pear andinOther
Pear and Other Rosaceae
Rosaceae SpeciesSpecies
A neighbor-joining (NJ) phylogenetic tree was constructed with the Mega-X program
A neighbor-joining
using the(NJ) phylogenetic
CAMTA proteins of tree was constructed
Arabidopsis, tomato, and with
eightthe Mega-X
Rosaceae program
species (Figure 2).
using the CAMTAThe proteins
total 59ofCAMTA
Arabidopsis,
proteinstomato,
of the and eight Rosaceae
ten species species (Figure
were subsequently divided into 2). five
The total 59 CAMTA proteins
subgroups: of the
Group ten species
I–Group V. The were subsequently
distribution in the fivedivided
subgroups into
wasfive sub- in
as follows:
European pear, one protein was in Group I, two in Group
groups: Group I–Group V. The distribution in the five subgroups was as follows: in Euro- II, two in Group III, two in Group
IV, and two in Group V; in Chinese white pear, two proteins were in Group I, two in Group
pean pear, one protein was in Group I, two in Group II, two in Group III, two in Group
II, one in Group III, two in Group IV, and two in Group V. The difference in the group
IV, and two in Group V; in ofChinese
distribution white
the proteins pear,between
observed two proteins
Europeanwere in Group
and Chinese pear I, twobeindue to
might
Group II, one in Group III, twobetween
the divergence in Group IV, species.
the two and two Inin Group
apple, V. Thebelonged
one protein difference in theI, two
to Group
group distributiontoofGroup
the proteins observed
II, one to Group between
III, two to Group European
IV, and two and Chinese
to Group pear
V. The might of
distribution
CAMTA proteins of Japanese apricot and black raspberry
be due to the divergence between the two species. In apple, one protein belonged to Group was the same, with one protein in
Group I, one in Group II, one in Group III, 0 in Group IV, and one in Group V. In strawberry,
I, two to Group II, one to Group III, two to Group IV, and two to Group V. The distribution
there was one protein in Group I, 0 in Group II, one in Group III, 0 in Group IV, and one
of CAMTA proteins of Japanese
in Group apricot
V; in peach, thereand
wasblack
one inraspberry
Group I, one was the same,
in Group with
II, one one pro-
in Group III, one in
tein in Group I, one in Group
Group IV, andII,
oneone in Group
in Group V; in III, 0 in
sweet Group
cherry, 0 in IV, andI, one
Group in Group
1 in Group II, oneV.inInGroup
strawberry, there was one
III, one in protein
Group IV, inand
Group
one in I, Group
0 in Group II, one in Group
V (Supplementary File S7:III, 0 inS3).
Table Group
We found
IV, and one in Group V; in peach, there was one in Group I, one in Group II, one in Group they
CAMTA proteins from different species clustered into the same subgroup, suggesting
may have similar functions.
III, one in Group IV, and one in Group V; in sweet cherry, 0 in Group I, 1 in Group II, one
in Group III, one in Group IV, and one in Group V (Supplementary File S7: Table S3). We
found CAMTA proteins from different species clustered into the same subgroup, suggest-
ing they may have similar functions.
Horticulturae 2023, 9, 467 6 of 19
Horticulturae 2023, 9, x FOR PEER REVIEW 6 of 18

Figure 2. Phylogenetic tree and distribution of conserved motifs across the CAMTA protein family
Figure 2. Phylogenetic tree and distribution of conserved motifs across the CAMTA protein family in
in eight Rosaceae species. The phylogenetic tree of the CAMTA protein family is constructed by
eight Rosaceae species. The phylogenetic tree of the CAMTA protein family is constructed by iqtree
iqtree using the neighbor-joining (NJ) method with 1000 bootstraps. A total of 20 motifs are pre-
using
dictedtheusing
neighbor-joining
the MEME tool. (NJ) Among
method these,
with 1000 bootstraps.
yellow branches A indicate
total of 20 motifsI; are
Group pinkpredicted
branches using
indi-
the MEME tool. Among these, yellow branches indicate Group I; pink branches
cate Group II; blue branches indicate Group III; gray branches indicate Group IV; and brown indicate Group II;
blue branches indicate Group III; gray branches indicate Group IV; and brown
branches indicate Group V. Blue star, yellow star, green star and pink star indicate the CAMTA branches indicate
proteins,
Group respectively,
V. Blue star, yellow instar,
Arabidopsis,
green starP.and
bretschneideri, P. communis,
pink star indicate the CAMTAand S.proteins,
lycopersicum. Pink tick,
respectively, in
green tick, purple tick, and blue tick indicate CAMTA proteins, respectively, in F.
Arabidopsis, P. bretschneideri, P. communis, and S. lycopersicum. Pink tick, green tick, purple tick,vesca, P. mume,andP.
persica,
blue tick and R. occidentalis.
indicate The orange
CAMTA proteins, circles and
respectively, insquares
F. vesca,represent
P. mume, P.CAMTA proteins
persica, and in M. domestica
R. occidentalis. The
and P. avium.
orange circles and squares represent CAMTA proteins in M. domestica and P. avium.

3.3.
3.3.Conserved
ConservedMotif
Motifand
andGene
GeneStructure
StructureAnalysis
AnalysisofofCAMTA
CAMTAFamily
FamilyininRosaceae
RosaceaeSpecies
Species
WeWeidentified
identified2020conserved
conservedmotifsmotifsininthetheCAMTA
CAMTAproteinsproteinsamong
amongten tenspecies
speciesandand
visualized
visualizedtheir theiramino
aminoacidacidcompositions
compositions(Supplementary
(SupplementaryFile FileS1:
S1:Figure
FigureS1) S1)using
usingthethe
MEME tool. The structures and locations of the conserved motifs
MEME tool. The structures and locations of the conserved motifs in each subgroup were in each subgroup were
nearly
nearlyidentical,
identical,indicating
indicatingthatthatthe
theproteins
proteinsfrom fromthethesame
samesubgroup
subgrouplikely
likelyhave
havesimilar
similar
functions.
functions. Based
Based on onmotif
motifanalysis
analysisandandphylogenetic
phylogenetictree treeresults,
results,weweobserved
observedthat thatthe
the
domains
domainsofofthe theCAMTA
CAMTAproteins
proteinsininthetheeight
eightRosaceae
Rosaceaespecies
specieswere
wererelatively
relativelyconserved;
conserved;
ininfact,
fact,allallproteins
proteins except
exceptPavCAMTA3
PavCAMTA3 contained
contained Motif 1, and
Motif all proteins
1, and had had
all proteins Motif 3. As3.
Motif
confirmed
As confirmed by the results of multi-sequence comparisons (Supplementary File S2: S2),
by the results of multi-sequence comparisons (Supplementary File S2: Figure Fig-
Motif
ure S2),1 (CG-1)
Motif 1and Motif
(CG-1) 3 (TIG)
and Motif were
3 (TIG) twowerehighly
twoconserved functional
highly conserved domains
functional in all
domains
CAMTA proteinsproteins
in all CAMTA found infound
the eight Rosaceae
in the species considered
eight Rosaceae in our study.
species considered Interestingly,
in our study. In-
all proteins had a conserved domain consisting, in order,
terestingly, all proteins had a conserved domain consisting, in order, of Motifof Motif 4, Motif 5, 4,
Motif
Motif15,5,
and Motif
Motif 15, 4,
andindicating
Motif 4, that this may
indicating be this
that a highly
may conserved
be a highlyfunctional
conserveddomain in CAMTA.
functional domain
The composition of conserved motifs in the same subgroup was
in CAMTA. The composition of conserved motifs in the same subgroup was almost iden- almost identical, such as
intical,
Group I, which did not contain Motif 11; Group II did not contain
such as in Group I, which did not contain Motif 11; Group II did not contain Motif Motif 13; Group V
contained Motif 18, which suggested that the proteins in the same
13; Group V contained Motif 18, which suggested that the proteins in the same subgroups subgroups may have
similar
may have structures
similarand functions.
structures and Conserved
functions. motif analysismotif
Conserved provided reliable
analysis evidence
provided and
reliable
strongly supported the results of the clustering in the phylogenetic
evidence and strongly supported the results of the clustering in the phylogenetic tree. tree.
InInorder
order toto clarify CAMTA gene
clarify CAMTA genestructures
structuresand andexplore
explore their
their structural
structural diversity,
diversity, we
we analyzed the intron and exon components by aligning their genomic sequences with
analyzed the intron and exon components by aligning their genomic sequences with the
9, x FOR PEER REVIEW 7 of 18
Horticulturae 2023, 9, 467 7 of 19

CDS of eight Rosaceae CAMTA

the CDS genes.
of eight As shown
Rosaceae CAMTAin Figure
genes. As3,shown
the exon number
in Figure ofexon
3, the the 46
number of
CAMTA genes ranged CAMTA
the 46from genes ranged from
10 (PavCAMTA3, 10 (PavCAMTA3,
sweet cherry) to 24sweet cherry) to 24
(FvCAMTA1, (FvCAMTA1,
straw-
strawberry),
berry), suggesting that suggesting
the CAMTA genethat the CAMTA
structures in gene structures in
the Rosaceae the Rosaceae
family family are diverse.
are diverse.

Figure 3. Phylogenetic relationship and gene structure of the CAMTA gene family in eight Rosaceae
Figure 3. Phylogenetic relationship and gene structure of the CAMTA gene family in eight Rosaceae
species. (A) The phylogenetic
species. (A)tree
Theisphylogenetic
constructed byisiqtree
tree usingbythe
constructed neighbor-joining
iqtree (NJ) method
using the neighbor-joining (NJ) method
with 1000 bootstraps.with
(B) 1000
Intron–exon structures of CAMTA genes in eight species.
bootstraps. (B) Intron–exon structures of CAMTA genes in eight species.

3.4. Chromosomal Distribution and Synteny Analysis of CAMTA Genes in Pear and Other
Rosaceae Species
We analyzed the genomic distribution of CAMTA genes on the chromosomes of eight
species. Six of the nine PbrCAMTA genes in Chinese white pear were unevenly distributed
throughout four chromosomes (Figure 4A). PbrCAMTA1 was on chromosome 5, while
PbrCAMTA2, PbrCAMTA3, and PbrCAMTA4 were co-located on chromosome 13.
Horticulturae 2023, 9, 467 8 of 19

3.4. Chromosomal Distribution and Synteny Analysis of CAMTA Genes in Pear and Other
Rosaceae Species
We analyzed the genomic distribution of CAMTA genes on the chromosomes of eight
species. Six of the nine PbrCAMTA genes in Chinese white pear were unevenly distributed
throughout four chromosomes (Figure 4A). PbrCAMTA1 was on chromosome 5, while Pbr-
CAMTA2, PbrCAMTA3, and PbrCAMTA4 were co-located on chromosome 13. PbrCAMTA5
was on chromosome 15, and PbrCAMTA6 was on chromosome 16. The remaining three
PbrCAMTA genes were mapped to different scaffold contigs. We analyzed the collinearity
relationships of CAMTA genes between Chinese white pear and the other seven Rosaceae
species in order to better understand the evolutionary mechanism of CAMTA genes. As
shown in Figure 4A, we observed good collinearity in that nine PbrCAMTA genes in Chinese
white pear separately corresponded to 14 homologous gene pairs in apple, nine pairs in
European pear, ten pairs in peach, five pairs in sweet cherry, eight pairs in Japanese apricot,
six pairs in black raspberry, and four pairs in strawberry. There were more collinear gene
pairs between Chinese white pear and European pear or Chinese white pear and apple,
indicating that these three species have close relationships, which is consistent with our
analysis. Here, we identified four homologous PbrCAMTA gene pairs (Figure 4B), which
contained eight PbrCAMTA genes (PbrCAMTA1-PbrCAMTA7, PbrCAMTA2-PbrCAMTA6,
PbrCAMTA4-PbrCAMTA9 and PbrCAMTA5-PbrCAMTA8), while PbrCAMTA3 was the only
gene with no collinear gene pair. Through further duplication event type analysis, we
found that eight PbrCAMTA genes all originated from a WGD/segmental duplication event,
while PbrCAMTA3 originated from a dispersed duplication event (Supplementary File S8:
Table S4).

3.5. Cis-Acting Elements Analysis in the Putative Promoter of CAMTA Genes in Rosaceae Species
The function and expression of a gene are related to the type of cis-acting element
contained in the upstream promoters. We identified a total of 6584 cis-acting elements
for 45 CAMTA gene promoters in the eight Rosaceae species considered. We selected
and clustered 12 varieties of important cis-acting elements for further analysis (Figure 5,
Supplementary File S9: Table S5): Abscisic acid (ABA)-responsive elements, Anaerobic-
induction-responsive elements, Defense-and-stress-responsive elements, Gibberellins (GA)-
responsive elements, Light-responsive elements, Methyl Jasmonate (MeJA)-responsive
elements, Salicylic acid (SA)-responsive elements, MYBHv1 binding sites, Zein-metabolism-
regulation-responsive elements, Low-temperature-responsive elements, and Meristem
expression regulatory elements. All Rosaceae CAMTA had between one (PavCAMTA3)
and twenty-two (RoCAMTA3) light-responsive elements, and 77.78% of the genes of all
CAMTAs had ABA-responsive elements. Interestingly, we noticed that 55.56% of the
PbrCAMTA genes of Chinese white pear had the zein metabolism-regulation-responsive
element, while only 11.11% of the PcpCAMTA genes of European pear had that element,
leading to divergence in the two pear species. The zein metabolism-regulation-responsive
element is reported to be associated with fruit softening [28]. Our findings indicate that
this element may have an effect on gene expression and may be involved in fruit maturity
phenotypes in Chinese white pear and European pear.
Horticulturae 2023, 9, 467 9 of 19

Figure 4. Synteny analysis in Chinese white pear. (A) Chromosomal distribution and synteny analysis
of pear and seven other species. The chromosomes of different species are shown in different color
blocks. Short lines located on different blocks represent the approximate distribution of each CAMTA
gene. Long lines denote the details of collinear gene pairs between Chinese white pear and the
other seven species. (B) Gene location and synteny analysis of PbrCAMTA genes. The homologous
genes of the PbrCAMTA family are identified using MCScanX software, and the circle picture was
plotted using Circos software (version 0.69.2). Red short lines indicate the position distribution
1 of PbrCAMTA genes on 17 pear chromosomes. Green curves show the collinear gene pairs of the
PbrCAMTA gene family.
 PbrCAMTA gene family.

3.5. Cis-Acting Elements Analysis in the Putative Promoter of CAMTA Genes in Rosaceae
Species
Horticulturae 2023, 9, 467 The function and expression of a gene are related to the type of cis-acting 10 of 19element

contained in the upstream promoters. We identified a total of 6584 cis-acting elements for
45 CAMTA gene promoters in the eight Rosaceae species considered. We selected and
3.6. Transcriptome
clustered and qRT-PCR
12 varieties Analysiscis-acting
of important of PbrCAMTA Genes for further analysis (Figure 5, Sup-
elements
We analyzed
plementary File the
S9: transcriptome
Table S5): Abscisicdata of six (ABA)-responsive
acid tissues (stem, leaf, petal, fruit, ovary,
elements, and
Anaerobic-induc-
sepal) of ‘Dangshansuli’
tion-responsive elements,to obtain the expression patterns of PbrCAMTAs.
Defense-and-stress-responsive The RPKM(GA)-re-
elements, Gibberellins
values represent
sponsive the expression
elements, Light-responsive PbrCAMTAs,
levels of elements, and the
Methyl heatmap(MeJA)-responsive
Jasmonate result is shown ele-
in Figure 6A. We considered genes not to be expressed for RPKM values <= 1 and genes
ments, Salicylic acid (SA)-responsive elements, MYBHv1 binding sites, Zein-metabolism-
to have a low expression when 1 < RPKM < 3. PbrCAMTA genes were clustered into two
regulation-responsive elements, Low-temperature-responsive elements, and Meristem
classes based on the transcriptome data analysis: Class I included PbrCAMTA1, 2, 6, 7; Class
IIexpression regulatory 4,
included PbrCAMTA3, elements.
5, 8, 9. WeAll Rosaceae
found that theCAMTA
four geneshad(PbrCAMTA1,
between one2, (PavCAMTA3)
6, 7) in
and twenty-two (RoCAMTA3) light-responsive elements, and 77.78%
Class I were highly expressed in all six tissues, meaning they may have different functions of the genes of all
in different tissues, and they may play important roles in plant growth and development. of the
CAMTAs had ABA-responsive elements. Interestingly, we noticed that 55.56%
OnPbrCAMTA
the contrary,genes
ClassofIIChinese
genes may white pearonly
function hadinthe zein metabolism-regulation-responsive
specific tissues, such as PbrCAMTA5
element,
and while only
PbrCAMTA8 11.11%
in leaves, of the PcpCAMTA
PbrCAMTA3 in stems, genes of European
and PbrCAMTA9, pearwas
which hadhighly
that element,
expressed in the petal tissue. In addition, we selected four PbrCAMTA genes
leading to divergence in the two pear species. The zein metabolism-regulation-responsive (PbrCAMTA2,
4,element
7, 8) to verify their expression
is reported levels in
to be associated six tissues
with of ‘Dangshansuli’
fruit softening [28]. Our by quantitative
findings indicate that
RT-PCR experiment. As shown in Figure 6B, four genes had specific expression
this element may have an effect on gene expression and may be involved in fruit maturity patterns,
which were largely consistent with our transcriptome data demonstrating its reliability.
phenotypes in Chinese white pear and European pear.

Figure5.5.Cis-acting
Figure Cis-actingelements
elements
inin putative
putative promoters
promoters (2000
(2000 bp bp upstream)
upstream) of PbrCAMTA
of PbrCAMTA genes
genes in in Chi-
nese white pear. (A) Distribution of cis-acting elements on putative promoters of PbrCAMTA genes.
Chinese white pear. (A) Distribution of cis-acting elements on putative promoters of PbrCAMTA
genes. The phylogenetic tree of the PbrCAMTA genes family was constructed by using iqtree using
the neighbor-joining (NJ) method and 1000 bootstraps. (B) The number of cis-acting elements on
putative promoters of PbrCAMTA genes. A total of twelve cis-acting elements are investigated,
including: (I) ABA-responsive elements, (II) Anaerobic induction-responsive elements, (III) Auxin-
responsive elements, (IV) Defense-and-stress-responsive elements, (V) GA-responsive elements,
(VI) Light-responsive elements, (VII) MeJA-responsive elements, (VIII) Salicylic-acid-responsive
elements, (IX) MYBHv1 binding sites, (X) Zein metabolism-regulation-responsive elements, (XI) Low-
temperature-responsive elements, and (XII) Meristem expression regulatory elements.
Horticulturae 2023, 9, 467 11 of 19

In order to investigate the expression patterns of PbrCAMTA genes during pear fruit
development in different pear cultivars, we analyzed the fruit transcriptome data of six
major cultivars: ‘Hosui’, ‘Kuerlexiangli’, ‘Nanguoli’, ‘Starkrimson’, ‘Yali’ and ‘Dangshan-
suli’. The results (Figure 7A) showed that nine PbrCAMTA genes had specific expression
trends. Among these genes, PbrCAMTA1, 2, 6, and 7 were highly expressed in six peri-
ods of all cultivars; PbrCAMTA3, 4, 5, 8, and 9 had relatively high levels of expression
in some periods. The diversity of PbrCAMTAs expression patterns in fruits by qRT-PCR
(Figure 7B) demonstrates that these genes may play different roles in fruit development.
Horticulturae 2023, 9, x FOR PEER REVIEW 11 of 18
This observation lays a significant foundation for future research on gene functions.

Figure 6. Heatmap and qRT-PCR analysis of expression levels of PbrCAMTA genes in tissues. (A)
Figure 6. Heatmap and qRT-PCR analysis of expression levels of PbrCAMTA genes in tissues.
Heatmap of the expression level (log2(RPKM+0.01)) of nine PbrCAMTA genes in six different tissues
(A) Heatmap of the expression level (log2 (RPKM+0.01)) of nine PbrCAMTA genes in six different
(leaf, petal, fruit, stem, ovary, and sepal) in ‘Dangshansuli’. The heatmap is plotted by pheatmap;
tissues (leaf, petal,
red indicates highfruit, stem, ovary,
expression, and and sepal)
blue in ‘Dangshansuli’.
indicates The heatmap
low expression. is plotted
The color scale atby pheatmap
the top right
represents RPKM values normalized by log2. (B) qRT-PCR analysis of four PbrCAMTA genes in six
different tissues. The x-axis represents six different tissues (stem, ovary, petal, sepal, fruit, and leaf),
and the y-axis represents relative expression levels of the PbrCAMTA genes. Error bars indicate
three technical replicates derived from one bulked biological replicate.
Horticulturae 2023, 9, 467 12 of 19

red indicates high expression, and blue indicates low expression. The color scale at the top right
represents RPKM values normalized by log2. (B) qRT-PCR analysis of four PbrCAMTA genes in six
different tissues. The x-axis represents six different tissues (stem, ovary, petal, sepal, fruit, and leaf),
Horticulturae 2023, 9, x FOR PEER REVIEW
and the y-axis represents relative expression levels of the PbrCAMTA genes. Error bars indicate 12 three
of 18

technical replicates derived from one bulked biological replicate.

Figure 7. Heatmap and qRT-PCR analysis of expression levels of PbrCAMTA genes in different pe-
Figure 7. Heatmap and qRT-PCR analysis of expression levels of PbrCAMTA genes in different periods
riods of six pear cultivars. (A) Heatmaps of expression level analysis of PbrCAMTA genes at DAFB
of six pear cultivars. (A) Heatmaps of expression level analysis of PbrCAMTA genes at DAFB of six
of six different pear cultivars (‘Hosui’, ‘Kuerlexiangli’, ‘Nanguoli’, ‘Starkrimson’, and ‘Yali’). (B)
different
qRT-PCR pear cultivars
analysis of(‘Hosui’, ‘Kuerlexiangli’,
four PbrCAMTA genes‘Nanguoli’,
(PbrCAMTA2,‘Starkrimson’,
PbrCAMTA4,and ‘Yali’). (B) qRT-PCR
PbrCAMTA7, and
analysis of four PbrCAMTA genes (PbrCAMTA2, PbrCAMTA4, PbrCAMTA7, and PbrCAMTA8)
PbrCAMTA8) in fruit tissues of ‘Dangshansuli’ at different DAFB. The x-axis represents six different in fruit
tissues ofincluding
tissues, ‘Dangshansuli’
stem, at different
ovary, DAFB.
petal, sepal,The x-axis
fruit, and represents
leaf, and thesixy-axis
different tissues, relative
represents including stem,
expres-
sion levels
ovary, of PbrCAMTA
petal, sepal, fruit, andgenes. Error
leaf, and thebars indicate
y-axis threerelative
represents technical replicates
expression derived
levels from one
of PbrCAMTA
bulked
genes. biological
Error replicate.
bars indicate three technical replicates derived from one bulked biological replicate.

3.7. Effects of PbrCAMTA Expression Manipulation

Based on transcriptome data and analysis, we hypothesized that PbrCAMTA2 could
be related to fruit ripening. To validate the function of PbrCAMTA2, we transiently ex-
pressed PbrCAMTA2 in ‘Zaosu’ pear fruits at 110 DAFB by injecting Agrobacterium con-
Horticulturae 2023, 9, 467 13 of 19

3.7. Effects of PbrCAMTA Expression Manipulation

Based on transcriptome data and analysis, we hypothesized that PbrCAMTA2 could be
related to fruit ripening. To validate the function of PbrCAMTA2, we transiently expressed
PbrCAMTA2 in ‘Zaosu’ pear fruits at 110 DAFB by injecting Agrobacterium13containing
Horticulturae 2023, 9, x FOR PEER REVIEW of 18 the
p1300-35S:PbrCAMTA2-GFP construct vector. We injected the empty vector as a control
at the symmetrical position in the treated pear fruits (Figure 8A). Seven days after the
injection,
as a controlthe pear
at the fruit tissues
symmetrical wereincollected
position the treatedtopear
measure the firmness
fruits (Figure and
8A). Seven extract the
days
RNA. Weinjection,
after the found that the tissues
the pear thatwere
fruit tissues overexpressed PbrCAMTA2
collected to measure had better
the firmness firmness than
and extract
the RNA.
those We found
injected thatempty
with the the tissues that overexpressed
vectors (Figure 8B). APbrCAMTA2
quantitative had better firmness
RT-PCR experiment was
than those injected with the empty vectors (Figure 8B). A quantitative
used to investigate PbrCAMTA2 and the other genes involved in fruit ripening, RT-PCR experiment including,
was used toendotransglucosylase
xyloglucan investigate PbrCAMTA2 (XET) and the[29],
otherand
genes involved in fruit ripening, in-
1-Aminocyclopropane-1-Carboxylate
cluding, xyloglucan endotransglucosylase (XET) [29], and 1-Aminocyclopropane-1-Car-
Oxidase (ACO) [30] and β-galactosidase (TBG) [31] The results showed that PbrCAMTA2
boxylate Oxidase (ACO) [30] and β-galactosidase (TBG) [31] The results showed that
was overexpressed over 23-fold in pear fruits compared to the tissues injected with the
PbrCAMTA2 was overexpressed over 23-fold in pear fruits compared to the tissues in-
empty vector
jected with the(Figure 8C). We
empty vector also8C).
(Figure observed
We alsothat the expressions
observed of TBG, of
that the expressions XET,
TBG,and ACO
were lower in fruits with overexpressed PbrCAMTA2, indicating
XET, and ACO were lower in fruits with overexpressed PbrCAMTA2, indicating that that PbrCAMTA2 may
delay pear fruit
PbrCAMTA2 mayripening byfruit
delay pear ripeningTBG,
regulating XET, and
by regulating ACO.
TBG, XET, and ACO.

Figure 8. Transient assays by overexpressing PbrCAMTA2 in ‘Zaosu’ pear fruit 110 DAFB. (A) Phe-
Figure 8. Transient assays by overexpressing PbrCAMTA2 in ‘Zaosu’ pear fruit 110 DAFB. (A) Phenotypes
notypes of pear fruits. Images were taken five days after agro-infiltration. (B) The firmness of fruits.
Horticulturae 2023, 9, 467 14 of 19

of pear fruits. Images were taken five days after agro-infiltration. (B) The firmness of fruits. The
mean separations are conducted by t-test in the GraphPad Prism 8. Asterisks indicate that significant
differences existed (** p < 0.01, *** p < 0.001 and **** p < 0.0001). (C) qRT-PCR analysis of PbrCAMTA,
ACO, XET, and TBG genes in different experimental groups. The pink columns represent PbrCAMTA2,
and the blue columns represent the empty vector. Error bars indicate three technical replicates derived
from one bulked biological replicate.

4. Discussion
4.1. Identification and Phylogenetic Analysis of the CAMTA Family in Eight Rosaceae Species
We identified a total of 46 CAMTA proteins from eight species of the Rosaceae family,
nine of which were in Chinese white pear, nine in European pear, eight in apple, four in
black raspberry, three in strawberry, four in Japanese apricot, four in sweet cherry, and
five in peach. Additionally, two model plants were introduced to assist in the CAMTA
genes classification. Finally, the 46 CAMTA proteins were clustered into five subgroups
(Figures 1 and 2). Our results are similar to the clustering results obtained for other
species, as ten VvCAMTA genes are classified into four groups in grapes [11], and nine
CitCAMTA genes are clustered into five groups in citrus [16]. The distribution of the
proteins from each Rosaceae species was various. Proteins in the same subgroup often
have similar functions. In addition, based on the results of the multi-sequence comparison
and conservative domain analysis, 98.30% of the CAMTA members had a conserved DNA-
binding domain (CG-1), consisting of approximately 130 amino acids, which is involved
in regulating the transcription and expression of genes associated with ethylene, abscisic
acid, and the light-responsive element [32]. All of the CAMTA genes had a transcription
factor immunoglobulin domain (TIG), which is involved in the non-specific binding of
various transcription factors, initiating the dimerization of proteins [10]. CAMTA proteins
of different subgroups all contained those two motifs, suggesting that the Rosaceae CAMTA
family is a highly conserved transcription factor family.
In addition, gene structure analysis showed most CAMTA genes (87%) contained
9–13 introns (Figure 3), similar to the number of introns in CAMTA genes in other species:
10–13 in wheat [33], 9–12 in Gossypium [15], 6–12 in citrus [16], and 10–12 in banana [17].
We observed the physical properties of the CAMTA protein sequences of eight Rosaceae
species (Supplementary File S6: Table S2); the results showed that the CAMTA protein
family generally had longer sequences (854–2065 amino acids) and larger protein molecular
masses (96–233 kD), much higher than the other gene families in Rosaceae, such as the
ADH protein family, which has a length ranging from 300 to 887 amino acids and an MW
ranging from 32.32 to 69.83 kD [34].

4.2. Evolutionary Mechanism and Good Collinearity Relationships between Pear and Other Seven
Species of the Rosaceae Family
Gene duplication events can be mainly traced to five origins: genome-wide duplica-
tion (WGD), tandem (TD), proximal (PD), transposed (DD), and dispersed duplications
(DSDs) [35]. Many gene families have expansion events in Rosaceae [36]. Previous research
has shown that there are two whole-genome duplication (WGD) events that occur in pear
and apple, with a recent WGD event taking place at 30–45 MYA and an ancient WGD
event that occurred at ~140 MYA [23,37]. In this research, eight of the nine PbrCAMTA
genes in pear resulted from WGD/segmental duplication, and one remaining PbrCAMTA
gene resulted from dispersed duplication (Supplementary File S8: Table S4), indicating
that the WGD/segmental event was the main driving force in the evolution of CAMTA
genes in Chinese white pear. Researchers have also proved that a similar situation occurred
in other gene families in pear. The SWEET gene family, for instance, is found to have
WGD/segmental duplication as the primary evolutionary mechanism [38].
Between Chinese white pear and the other seven Rosaceae species, we detected good
collinearity relationships (Figure 4A). We found that the most collinear gene pairs (14) were
Horticulturae 2023, 9, 467 15 of 19

shared between Chinese white pear and apple. Considering that both pear and apple have
undergone a recent WGD event, they are expected to have a closer relationship. Only ten
collinear pairs shared between Chinese white pear and European pear may be due to the
interspecific divergence that arose during evolution. Among the nine PbrCAMTA genes,
PbrCAMTA6 had the most collinear gene pairs (13) across seven species, indicating that
PbrCAMT6 may have evolved from ancestral pear genes. In addition, PbrCAMTA3 shared
only five collinear gene pairs with the other seven species, probably due to the dispersed
duplication event that caused conserved domain variation during evolution.

4.3. Cis-Acting Element Analysis of CAMTA Genes

Zein is a natural storage protein, and researchers have discovered that zein coatings
can delay the ripening process of mango by controlling the activity of softening enzymes,
such as pectin methyl esterase and cellulase [28]. In pears, we found that 55.56% of
PbrCAMTAs had zein metabolism-regulation-responsive elements in their promoters in
Chinese white pear, while only 11.11% of PcpCAMTAs had the element in P. communis.
There are different maturity phenotypes between the two pear cultivars; the fruits of
P. communis have a post-ripening process and usually have a good flavor after post-harvest
storage, while P. bretschneideri fruits can be eaten immediately after harvest [25]. Based on
this, we speculated that the zein metabolism-regulation-responsive element regulates pear
fruit softening divergence between Chinese white pear and European pear cultivars by
participating in differential expressions of CAMTA genes.
Most of the CAMTA-promoter cis-acting elements of Rosaceae species contained
ABA-responsive elements, especially in pears, with the ABRE element being present in all
PbrCAMTA genes. The ABRE element is known to act as a signal response cis-acting element
to conduct Ca2+ signals, and CAMTA transcription factors in pear may be associated with
responses to ABA and environmental stress [39]. PbrCAMTAs may participate in the
Abscisic acid (ABA) metabolic pathway through the ABA cis-acting element in order to
regulate pear fruit ripening. In addition, previous studies have found that hormones, such
as Gibberellins (GA) and Methyl Jasmonate (MeJA), are related to fruit firmness. Gibberellin
could delay maturation by upregulating auxin [40], while MeJA could accelerate fruit
softening by promoting the expression of XTH1 and EG1 [41].

4.4. Expression Analysis and Functional Prediction of Candidate PbrCAMTA Genes

CAMTA family members have proven to be involved in fruit softening, signal transduction,
and abiotic and biotic stress response during the development of many plants [8,12,16,17,42].
However, there have only been a few studies on CAMTA family members in relation to
pear. Our research on PbrCAMTAs expression patterns has the potential to provide solid
bases for further analysis. In transcriptome analysis, nine PbrCAMTA genes showed di-
verse expression patterns in different tissues and during different fruit development stages
(Figures 6A and 7A). From the results of qRT-PCR (Figures 6B and 7B), we observed that
during the fruit development of ‘Dangshansuli’ pear, the expression of most of the Pbr-
CAMTA genes was higher before fruit development 36 DAFB, indicating that PbrCAMTAs
may affect the development of young fruits. During 80 DAFB-167 DAFB, the expression
of PbrCAMTAs showed an increasing trend, and a similar trend has been detected in
tomatoes [8]. PbrCAMTAs are likely to be involved in fruit development and softening.
We found that most PbrCAMTA genes were expressed at high levels in leaves, sug-
gesting those PbrCAMTA genes may play crucial roles in pear leaf tissues and plant stress
response [9,43]. Proteins in the same subgroup often have similar functions, which might
be traced back to a common ancestral protein [36]. PbrCAMTA8 was classified in Group
V, together with AtCAMTA3, which has been reported to negatively regulate salicylic
acid-mediated plant immunity [22], suggesting that the PbrCAMTA8 gene might function
similarly to AtCAMTA3 and could be involved in SA metabolism, negatively regulating
immunity in pear. Fruit setting is the developmental transition from the ovary to the
young fruit [44]. We found that PbrCAMTA2 had a higher expression level in the ovary.
Horticulturae 2023, 9, 467 16 of 19

PbrCAMTA2 may be related to fruit setting by affecting the development of the ovary and
that of the young fruit. Additionally, we noticed that PbrCAMTA2 had a trend of high
expression in the early stages of development and low expression in the ripening stage.
Previous studies have shown that CAMTA is involved in fruit softening [8]. Therefore, we
speculated that PbrCAMTA2 might be involved in the softening of pear fruits. This conjec-
ture was confirmed by transient transformation results (Figure 8B,C). It is believed that fruit
softening is due to ethylene-mediated enzymatic modifications of the cell wall. Different
types of cell-wall-modifying enzymes, such as TBG and XET, are thought to play important
roles in the ripening process [5,45]. ACO is believed to participate in the de novo synthesis
of ethylene [7]. Based on these findings, we chose to consider these genes for further
research on fruit softening. Finally, our results indicated that overexpressing PbrCAMTA2
caused significant decreases in the expression levels of cell-wall-related genes: XET, TBG,
and ethylene-synthesis-related genes, such as ACO (Figure 8C). We performed a cis-acting
analysis of PbrACO, PbrXET, and PbrTBG putative promoters (2000 bp upstream) using
the PlantCARE website in order to explore possible relationships with PbrCAMTA2 further.
As shown in Figure S4 (Supplementary File S4), ABRE elements were widely found in the
promoter regions (2000 bp) of three genes. It has been reported that as the Ca2+ -dependent
transcription factor in plants, CAMTA may function as a link between Ca2+ signaling and
ABRE-related cis-elements [46,47]. Therefore, we speculated that PbrCAMTA2 regulates
downstream gene expression, such as softening-related enzymes (TBG, ACO, and XET), by
binding to ABRE elements. Therefore, we hypothesized that PbrCAMTA2 might influence
pear fruit ripening and extend the storage period of pears.

5. Conclusions
We identified 46 CAMTA genes in the genomes of eight Rosaceae species and stud-
ied their physicochemical properties. We clustered 59 CAMTA proteins from the eight
species and two plant model species into five subgroups. Duplication events analysis
proved that WGD/segmental duplication and dispersed duplication were the main driving
force in the expansion of the PbrCAMTA gene family. We identified four homologous
WGD gene pairs in the PbrCAMTA family. The promoters of nine PbrCAMTA genes con-
tained cis-acting elements related to light response, hormone response, and stress response.
Through transcriptome data and qRT-PCR experiments, we concluded that the expression
of PbrCAMTA is different in plant growth and in development. Transient transformation
assays demonstrated that PbrCAMTA2 could regulate fruit softening. Our results help
to clarify the biological function of PbrCAMTA genes in pear development (pear fruit
development and different tissues) and have a significant influence on our knowledge of
woody plant CAMTAs.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/horticulturae9040467/s1, Figure S1: Amino acid compositions of
20 conserved motifs of the CAMTA family in eight Rosaceae species; Figure S2: Multiple sequence
alignment of CAMTA protein family in ten species; Figure S3: Chromosomal distribution and Synteny
analysis of CAMTA genes; Figure S4: Cis-acting analysis of PbrACO, PbrXET, and PbrTBG putative
promoters (2000 bp upstream); Table S1: Primers chart for qRT-PCR and vector construction; Table S2:
Characteristics of CAMTA genes in eight Rosaceae species; Table S3: The number of CAMTA of
eight Rosaceae species in five subgroups of phylogenetic tree; Table S4: Duplication events types of
PbrCAMTA gene family; Table S5: Cis-acting elements heatmap in the putative promoter of CAMTA
genes in eight Rosaceae species.
Author Contributions: Conceptualization, J.Y. and B.S.; methodology, J.Y., B.S. and B.C.; software,
J.Y. and B.S.; validation, J.Y. and K.G.; formal analysis, J.Y. and K.G.; investigation, B.C.; data curation,
K.Z.; writing—original draft preparation, J.Y.; writing—review and editing, B.S. and J.L.; funding
acquisition, J.W. All authors have read and agreed to the published version of the manuscript.
Horticulturae 2023, 9, 467 17 of 19

Funding: This work was funded by the National Key Research and Development Program of China
(2021YFD1200200), the National Natural Science Foundation of China (31725024 and 31801835), the
Earmarked Fund for China Agriculture Research System (CARS-28), and the Earmarked Fund for
Jiangsu Agricultural Industry Technology System (JATS [2021]453).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets generated for this study are available upon request to the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.

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