































Highlights in BioScience
ISSN:2682-4043
DOI:10.36462/H.BioSci.202302

Research Article

Open Access

1 Department of Genetic Resources, Desert

Research Center (DRC), Cairo, Egypt.

* To whom correspondence should be
addressed: drmokhtarsaid@yahoo.com

Editor: Aladdin Hamwieh, International Center
for Agricultural Research in the Dry Areas
(ICARDA), Giza, Egypt.

Reviewer(s):
Clara R. Azzam, Cell Research Department, Field
Crops Research Institute, Agricultural Research
Center, Giza 12619, Egypt.

Fida Alo, International Center for Agricultural
Research in the Dry Areas (ICARDA), Beirut,
Lebanon.

Thoria Rashad Mohamed, Cytology and genetics,
Botany department, women faculty, Cairo, Egypt.

Received: July 10, 2023

Accepted: Sep 6, 2023

Published: September 15, 2023

Citation: Rizk MS. Unveiling the architecture
variability of coding regions in Prunus
persica: Identification and characterization
of key gene families impacting plant
development. 2023 Sep 15;6:bs202302

Copyright: © 2023 Rizk. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction
in any medium, provided the original author and
source are credited.
Data Availability Statement: All relevant data are
within the paper and supplementary materials.
Funding: The authors have no support or funding
to report.
Competing interests: The author declare that they
have no competing interests.

Unveiling the architecture variability of coding regions in Prunus
persica: Identification and characterization of key gene families
impacting plant development

Mokhtar Said Rizk 1
>< 

Abstract

This study aimed to investigate gene families related to Prunus persica (peach) and
their evolutionary relationships. Gene identification and sequence alignment methods
were used to characterize Homeobox, MADS-box, NAC, and Receptor-kinase-like
gene families. Gene expression data analysis was conducted to assess gene activity
and regulatory mechanisms in peach plant development. Gene identification involved
a local BLASTp analysis using protein sequences from Arabidopsis thaliana gene
families as queries against the peach proteome. Multiple sequence alignment and
phylogenetic tree construction were performed using ClustalW and iToL, respectively.
Gene expression analysis utilized the GEO2R online tool and focused on two datasets,
GSE71561 and GSE71460. Gene identification revealed 593 genes in total, with
varying gene counts and sequence lengths among the different families. Phylogenetic
analysis demonstrated distinct clusters within each gene family. Protein-protein
interaction analysis showed significant interconnectivity among Homeobox
transcription factors, while MADS-box and NAC gene families displayed lower
connectivity. Enrichment analysis highlighted developmental processes associated
with these gene families. Gene expression analysis provided insights into peach
fruit development, ripening, and postharvest changes, revealing differential gene
expression patterns and regulatory mechanisms. This study provides valuable insights
into the gene families related to Prunus persica, their evolutionary relationships, and
regulatory mechanisms. The identified genes and their functional characteristics
contribute to a better understanding of peach development and fruit phenotypes.
Further research is required to elucidate the implications of gene variations and
interactions in the studied system.

Keywords: Peach, Gene expression analysis, Gene families, MADS-box, NAC gene family,

Receptor-kinase-like

Introduction
Peaches, scientifically known as Prunus persica, are fruit trees belonging to the rose family

(Rosaceae) and are cultivated in warmer temperate regions of both the Northern and Southern hemi-

spheres [1]. They are widely enjoyed fresh and are commonly used in baking, such as in pies and

cobblers. Canned peaches are also a popular staple in many areas. Varieties with yellow flesh are

particularly notable for their high vitamin A content [2]. Compared to some other fruit trees, peach

trees have a relatively shorter lifespan. In certain regions, orchards are replanted every 8 to 10

years, while in others, trees may continue to produce satisfactorily for 20 to 25 years or even longer,

depending on their resistance to diseases, pests, and winter-related damage [3].

Countries such as Spain, Italy, the United States, Chile, and Australia consider peach is as

highly significant Prunus specie. The European Union holds the second position worldwide in peach

production, with China being the only country ahead. Between 2018 and 2020, the EU averaged an

annual production of 3612,000 tons, while the harvested area for peaches in 2019 encompassed

206,660 hectares. Leading the rankings, Spain boasts 77,464 hectares of peach cultivation and

produces 1480,000 tonnes per year, closely followed by Italy and Greece [4].

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Rizk, 2023 Identifying and characterizing key gene families influencing Prunus persica plant development

Homeobox genes, MADS-box genes, NAC genes, and re-
ceptor -kinase-like genes are all involved in the regulation of
gene expression and play important roles in the development
and growth of organisms. Homeobox genes, MADS-box genes,
NAC genes, and receptor-kinase-like genes are crucial players in
gene expression regulation and exert significant roles in organ-
ismal development and growth [5; 6]. Specifically, homeobox
genes involved in floral organ development have been found to
interact with MADS-box genes, which in turn govern the expres-
sion of a wide array of genes responsible for flower development
and organ identity, including other MADS-box genes and home-
obox genes [7]. On the other hand, NAC genes exhibit interac-
tions with both homeobox genes and MADS-box genes, thereby
influencing their expression and contributing to the regulation of
plant development and stress responses [8; 9].

Receptor-kinase-like genes represent a diverse gene group
encoding receptor-like kinases (RLKs) or receptor-like proteins,
and some RLKs have demonstrated interactions with transcrip-
tion factors, including members of the MADS-box and NAC
gene families. These interactions are involved in the regulation
of gene expression and signaling pathways crucial for various
developmental processes and stress responses [10; 11]. Collec-
tively, the intricate network of interactions and regulatory mech-
anisms within these gene families contributes to the meticulous
control of gene expression and the coordination of developmen-
tal processes in organisms [12].

Studying these gene families, including Homeobox genes,
MADS-box genes, NAC genes, and receptor-kinase-like genes,
is of great importance in the field of peach genomics. These
gene families are involved in the regulation of gene expression
and play significant roles in the development and growth of or-
ganisms [13]. By understanding the functions and interactions
of these genes, researchers can gain insights into the molec-
ular mechanisms underlying peach development and improve
their understanding of the genetic factors that influence impor-
tant traits in peach [14].

The aim of this study is to accomplish the following ob-
jectives. Firstly, our aim is to identify and annotate the gene
family members, which include Homeobox genes, MADS-box
genes, NAC genes, and receptor-kinase-like genes, within the
peach genome. This objective will involve conducting a com-
prehensive genome-wide analysis and utilizing bioinformatics
tools to accurately identify and classify these gene family mem-
bers. Secondly, we aim to characterize the gene family mem-
bers to gain insights into their diversity and functional variations.
By investigating their unique features and roles in peach, we
can enhance our understanding of the genetic makeup of these
genes. Lastly, we intend to investigate the expression patterns of
the gene family members throughout plant development. This
will be achieved through high-throughput transcriptomic analy-
ses, such as RNA-sequencing, to capture the dynamic expression
profiles of these genes. By accomplishing these objectives, our
study aims to contribute to a deeper understanding of the genetic

characteristics, functional diversity, and expression dynamics of
Homeobox genes, MADS-box genes, NAC genes, and receptor-
kinase-like genes in peach.

Methods
Gene identification and sequence alignment

The methodology employed in this study aimed to investi-
gate the gene families related to Prunus persica (peach) and their
evolutionary relationships. The genome data used for the analy-
sis was obtained from Prunus persica NCBIv2 [15] , as provided
by the NCBI RefSeq database.

The gene families of interest included Homeobox, MADS-
box, NAC, and Receptor-kinase-like. The gene set comprised
a total of 593 genes, while Homeobox, MADS-box, NAC, and
Receptor-kinase-like gene families consisted of 91, 106, 94, and
302 genes, respectively. To further analyze these gene families,
information from Arabidopsis thaliana gene families was down-
loaded from the TIAR database, providing valuable reference
data [16].

To identify the corresponding gene family members in the
peach genome, a local BLASTp analysis was conducted. The
protein sequences from the Arabidopsis thaliana gene families
were used as queries against the peach proteome. The BLASTp
analysis was performed using the following parameters: "-outfmt
6 -evalue 1e-5 -num_threads 4" [17]. Genes that showed less
than 200 base pairs of match were subsequently filtered out from
further analysis.

To assess the evolutionary relationships among the identified
gene family members, a multiple sequence alignment was per-
formed using ClustalW, a widely used tool for aligning multiple
protein or nucleotide sequences [18]. The resulting alignment
was then utilized to construct a phylogenetic tree. The phylo-
genetic tree construction was accomplished using iToL (Interac-
tive Tree Of Life), an interactive web-based tool specifically de-
signed for visualizing and manipulating phylogenetic trees [19].
In the analysis of sequencing data, the Alignstatplot tool [20],
was employed to showcase both shared and distinct genomic re-
gions.

By implementing these methods, the study aimed to explore
the gene families related to Prunus persica and gain insights into
their evolutionary relationships. The genome data from Prunus
persica NCBIv2, combined with gene families information from
Arabidopsis thaliana, enabled the identification and characteri-
zation of the gene family members in peach. The BLASTp anal-
ysis, multiple sequence alignment, and phylogenetic tree con-
struction provided valuable information regarding the evolution-
ary history and relationships of these gene families.

Gene expression data analysis
Gene expression analysis was conducted using the GEO2R

online tool [21] to assess gene activity and regulatory mecha-
nisms in peach plant development. The researchers focused on
gene families such as Homeobox Transcription Factor, MADS-

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Rizk, 2023 Identifying and characterizing key gene families influencing Prunus persica plant development

box Gene, NAC Transcription Factor, and Receptor kinase-like
Gene families.

Two datasets, GSE71561 [22] and GSE71460 [23], were an-
alyzed. GSE71561 aimed to understand the molecular control
of peach fruit development, including interactions between seed
and mesocarp. It comprised 39 samples representing various
developmental stages, covering flower, seed, and mesocarp tis-
sues. Each sample had three biological replicates. Gene enrich-
ment analysis identified differentially expressed genes within the
same tissue stage, and the researchers examined tissue speci-
ficity versus stage specificity of the regulation.

GSE71460 focused on investigating the effects of gibberellic
acid application and cold storage on postharvest textural changes
in peaches, specifically woolliness. The dataset included 12
samples with four treatments, each having three biological repli-
cates. Approximately half of the investigated genes were ana-
lyzed, revealing significant differential expression. Gene ontol-
ogy and set enrichment analyses indicated the involvement of
cellular processes, developmental processes, and complex tran-
scriptional responses related to cell wall metabolism, hormone
metabolism, and signaling in response to cold storage and gib-
berellic acid.

By using the GEO2R online tool and analyzing these datasets,
the researchers gained insights into gene expression patterns and
regulatory mechanisms underlying peach fruit development, ripen-
ing, and postharvest changes. This study contributes to a better
understanding of the molecular processes governing these com-
plex phenotypes in peaches. Protein-protein interaction (PPI)
analysis was performed using the STRING database [24] and
enrichment analysis. The PPI network was visualized using the
Cytoscape program [25].

Results and Discussion
Gene identification and sequence variationa

Genes belonging to four distinct gene families were identi-
fied across peach genome (Figure 1). The results of the analysis
provided valuable insights into the gene counts and characteris-
tics of each family. The Homeobox transcription factor family
consisted of 52 genes, with a total length of 33,042 base pairs
(bp). Among these genes, the maximum sequence length ob-
served was 837 bp, while the minimum sequence length was
1,764 bp. For the MADS-box Gene family, a total of 16 genes
were identified, spanning a length of 4,522 bp. The maximum
sequence length observed in this family was 240 bp, and the
minimum sequence length was 349 bp. In the NAC transcrip-
tion factor family, a total of 42 genes were identified, with a
combined length of 15,903 bp. The maximum sequence length
observed within this family was 591 bp, while the minimum
sequence length was 862 bp (Figure 1). Finally, the Receptor
kinase-like Gene family exhibited the largest gene count, with
366 genes identified. These genes spanned a length of 300,904
bp. The maximum sequence length observed in this family was
626 bp, and the minimum sequence length was 556 bp (Figure

1). These results highlight the variations in gene counts and se-
quence lengths among the different gene families. They provide
valuable information about the diversity and characteristics of
genes within each family, shedding light on their potential roles
and functions in the studied system.

The multiple sequence alignment conducted in this study
provided valuable insights into the patterns of sequence variation
within the gene families analyzed (Figure 1). These variations
were evident through the presence of sequence gaps observed
in the alignments. It was noticeable that certain sequences ex-
hibited substantial portions of amino acid sequences that were
absent in others, indicating potential differences in gene func-
tion and underlying mechanisms (Figure 1). The presence of se-
quence gaps in the multiple sequence alignments suggests the oc-
currence of insertions or deletions in the gene sequences among
different members of the gene families. Such variations in se-
quence length and composition could have significant implica-
tions for the functionality and regulation of these genes. The
observed differences in amino acid sequences may lead to vari-
ations in protein structure and function, potentially influencing
their roles within the studied system [26]. By identifying these
sequence variations, this study contributes to a deeper under-
standing of the diversity and potential functional divergence within
the gene families analyzed. Further investigations into the spe-
cific functions and mechanisms associated with the unique se-
quence features observed could provide valuable insights into
the evolutionary dynamics and functional specialization of these
genes [27]. Additionally, the phylogenetic analysis based on the
sequence alignments revealed distinct clusters of genes within
each family, suggesting differences in gene functions and evolu-
tionary relationships (Figure 2).

The comparison between the results obtained in this study
and previous studies provides valuable insights into the gene
counts and characteristics of different gene families. A total of
73 homeobox-like genes in the grapevine genome were identi-
fied in a previous study [28]. The comparison between the re-
sults obtained in this study and previous studies provides valu-
able insights into the gene counts and characteristics of differ-
ent gene families. In the current study, our analysis identified
52 genes associated with Homeobox Transcription Factor Fam-
ily, suggesting variations in the composition and abundance of
Homeobox Transcription Factor genes between species or datasets.
Similarly, in the MADS-box Gene Family, our analysis identi-
fied only 16 genes, indicating potential disparities in the MADS-
box gene repertoire between the studied system and previous
studies in rice [29]. In relation to the NAC Transcription Fac-
tor Family, our analysis identified 42 genes, contrasting with
the 140 putative NAC or NAC-like genes identified in rice [30].
These contrasting gene counts suggest variations in the presence
or abundance of NAC genes between the studied system and rice.
The specific gene counts mentioned in the provided articles were
higher for the Homeobox Transcription Factor Family (73 genes)
and MADS-box Gene Family (75 genes) in grapes and rice, re-

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Rizk, 2023 Identifying and characterizing key gene families influencing Prunus persica plant development

Figure 1. Multiple sequence alignment of identified sequences from the Homeobox transcription factor family (A), MADS-box Gene family (B), NAC transcrip-

tion factor family (C), and Receptor kinase-like Gene family (D) in the peach genome. Sequences are color-coded, and gaps represent missing regions in the

alignment.

spectively. However, the gene counts obtained in this study may
be specific to the analysis or dataset used, and further research
is required to elucidate the reasons behind these variations and
their implications in the studied system.

Protein-protein interaction analysis
The analysis of the Homeobox transcription factor family

in the peach genome using protein-protein interaction (PPI) net-
work analysis revealed significant interconnectivity and poten-
tial functional relationships among the proteins (Figure 3). The

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Rizk, 2023 Identifying and characterizing key gene families influencing Prunus persica plant development

Figure 2. Phylogenetic trees illustrating the evolutionary relationships of identified sequences from the Homeobox transcription factor family (A), MADS-box

Gene family (B), NAC transcription factor family (C), and Receptor kinase-like Gene family (D) in the peach genome. The trees provide insights into the genetic

divergence and relatedness among the sequences within each gene family, highlighting their evolutionary history and potential functional similarities or distinc-

tions.

network consisted of 25 proteins and 61 interactions, indicat-
ing a complex regulatory network involved in peach develop-
ment. The average local clustering coefficient suggested clus-
tering of nodes, further supporting the notion of functional co-

operativity among the Homeobox transcription factors. The en-
riched Gene Ontology (GO) terms associated with the Home-
obox transcription factor family included various processes re-
lated to key developmental processes and organ polarity estab-

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Rizk, 2023 Identifying and characterizing key gene families influencing Prunus persica plant development

lishment in peach, such as determination of bilateral symme-
try, primary shoot apical meristem specification, xylem develop-
ment, meristem initiation, and polarity specification of the adax-
ial/abaxial axis. These findings highlight the important roles of
Homeobox transcription factors in orchestrating developmental
processes and regulating gene expression and cellular processes
in peach.

Several overlapping themes can be observed when compar-
ing these findings with published studies. A previous study fo-
cused on HD-Zip III homeobox genes and their involvement in
vascular differentiation [31]. They identified a new member of
the HD-Zip III genes, ZeHB-13, which exhibited restricted ex-
pression in the procambium. This finding aligns with the en-
riched GO term of xylem development identified in the peach
Homeobox transcription factor family. Both studies suggest that
Homeobox transcription factors play crucial roles in the regu-
lation of vascular development. The investigation of the TDIF
peptide signaling pathway and its impact on vascular stem cell
proliferation in Arabidopsis revealed the WOX4 homeobox gene
as a key target of the TDIF signaling pathway, promoting the pro-
liferation of procambial/cambial stem cells [32]. This finding is
consistent with the enriched GO term of meristem initiation and
the regulation of primary shoot apical meristem specification ob-
served in the peach Homeobox transcription factor family. Both
studies imply the involvement of Homeobox transcription fac-
tors in the regulation of stem cell proliferation and meristem
development. Furthermore, a study on homeobox genes asso-
ciated with lignification in bamboo shoots identified 115 home-
obox genes, including several classes such as the KNOX class,
that interacted with other transcription factors involved in lignin
synthesis [33]. This interaction network parallels the potential
functional relationships observed in the protein-protein interac-
tion network analysis of the peach Homeobox transcription fac-
tor family. Additionally, the upregulation of homeobox genes
in shoots as the height increased suggests their involvement in
developmental processes similar to the enriched GO terms iden-
tified in the peach study.

Similarly, the MADS-box gene family in the peach genome
was analyzed using PPI network analysis (Figure 3). The net-
work showed relatively low interconnectivity with 6 proteins
and 5 interactions. The average local clustering coefficient sug-
gested some tendency for nodes to cluster together. However,
the PPI enrichment analysis revealed functional associations or
co-regulation among the MADS-box gene family members. The
functional enrichment analysis identified processes such as Pollen
maturation, Regulation of pollen tube growth, Plant ovule devel-
opment, and Flower development associated with the MADS-
box gene family. These findings indicate their involvement in
important processes like pollen development, fertilization, and
flower formation in peach. In the context of these results, an in-
vestigation into the functional conservation of MADS-box genes
in Arabidopsis and rice pollen maturation was conducted [34].
The findings revealed that the disruption of specific MIKC-type

genes in rice resulted in severe defects in pollen maturation and
germination, providing supporting evidence for the involvement
of MADS-box genes in pollen development. Furthermore, a
study on MADS-box gene evolution beyond flowers identified
novel MADS-box genes in Arabidopsis, indicating that the evo-
lution of the MADS-box family involved rapid and simultane-
ous functional diversification in both vegetative and reproduc-
tive structures [35]. These findings align with the present re-
search, demonstrating functional associations or co-regulation
among the MADS-box gene family members. Additionally, a
study focused on the pineapple MADS-box gene family and its
evolutionary history emphasized the importance of understand-
ing the ancestral form of monocot flowers and their development,
further supporting the relevance of studying MADS-box genes
in different plant species [36].

In this study, we analyzed the NAC transcription factor fam-
ily in the peach genome using PPI network analysis and enrich-
ment analysis (Figure 3). The PPI network analysis revealed
a low level of connectivity and clustering within the NAC tran-
scription factor network, suggesting potential complexity in their
regulatory mechanisms. Additionally, the lack of significant en-
richment in the PPI analysis indicated sparse or transient interac-
tions among these proteins. However, the functional enrichment
analysis provided valuable insights into the involvement of NAC
transcription factors in diverse plant development processes.

Our findings are consistent with previous studies that have in-
vestigated the role of NAC transcription factors in specific plant
developmental processes. For instance, a study demonstrated
that the VASCULAR-RELATED NAC-DOMAIN7 (VND7) tran-
scription factor regulates the expression of genes required for
xylem vessel element formation in Arabidopsis. Multiple tran-
scription factors, including VND1-VND7, were identified as pu-
tative positive regulators of VND7 expression. This study high-
lights the importance of NAC transcription factors in xylem ves-
sel differentiation, which aligns with our functional enrichment
analysis showing their involvement in xylem vessel differentia-
tion and secondary cell wall biogenesis [37].

Another study discovered that VND-INTERACTING2 (VNI2),
a NAC domain transcription factor, negatively regulates xylem
vessel formation in Arabidopsis. VNI2 interacts with VND7
and other VND family proteins, acting as a transcriptional re-
pressor and repressing the expression of vessel-specific genes
regulated by VND7. This finding supports our findings of the
potential complexity in the regulatory mechanisms of NAC tran-
scription factors and their role in xylem vessel differentiation
[38]. Furthermore, a study investigated the function of XYLEM
NAC DOMAIN1 (XND1) in xylem differentiation. It revealed
that XND1 interacts with the RETINOBLASTOMA-RELATED
(RBR) protein and acts as a transcriptional repressor to inhibit
differentiation. These findings provide insights into the inhibitory
role of NAC transcription factors in xylem differentiation, which
aligns with our functional enrichment analysis showing their
involvement in secondary cell wall biogenesis [39]. In addi-

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Rizk, 2023 Identifying and characterizing key gene families influencing Prunus persica plant development

tion to their role in vascular development, NAC transcription
factors have been implicated in other plant developmental pro-
cesses. For example, a study investigated the involvement of
NAM genes, regulated by miR164, in floral-boundary morpho-
genesis in tomato. The NAM gene GOBLET was identified as
a key regulator of floral-boundary formation. Our functional en-
richment analysis revealed the involvement of NAC transcrip-
tion factors in shoot meristem specification and root cap develop-
ment, highlighting their role in various aspects of plant growth
and development [40]. Another study investigated the role of
CUP-SHAPED COTYLEDON1 (CUC1), a NAC transcription
factor, in the establishment of shoot organ boundaries in Ara-
bidopsis. It was found that CUC1 activates the expression of
LIGHT-DEPENDENT SHORT HYPOCOTYLS 4 (LSH4) and
LSH3, members of the ALOG gene family, in shoot organ bound-
ary cells. This study provides insights into the regulatory mech-
anism of shoot organ boundary establishment mediated by NAC
transcription factors [41].

The Receptor kinase-like gene family in the peach genome
was analyzed using PPI network analysis and enrichment anal-
ysis (Figure 3). The PPI network consisted of 151 nodes and
966 edges, indicating a dense network of protein interactions.
The network exhibited a high level of interconnectivity, suggest-
ing functional relevance and potential cooperativity among the
genes. The functional enrichment analysis provided insights into
the involvement of the Receptor kinase-like gene family in var-
ious developmental processes critical for plant growth and re-
sponse to environmental stimuli. The analysis also highlighted
molecular functions related to signal transduction and receptor
binding, emphasizing the importance of receptor-mediated sig-
naling pathways in peach. References to relevant scientific publi-
cations covered topics such as genetic incompatibility [42], self-
incompatibility signaling [43], protein secretion, gene duplica-
tions, and orthologous relationships [44], providing further in-
sights into the functional characteristics and evolutionary aspects
of the Receptor kinase-like gene family in peach. The enrich-
ment analysis included classifications from various databases
and ontologies, offering additional functional annotations and
domain information associated with the Receptor kinase-like gene
family, enhancing our understanding of their functional proper-
ties and characteristics.

Gene differential expression analysis
Gene expression analysis was performed to identify genes

highly associated with peach development using two different
datasets: GSE71460 and GSE71561. In GSE71460, which fo-
cused on the effects of gibberellic acid application and cold stor-
age on postharvest textural changes in peaches, several impor-
tant genes were identified. These included SKP1-like, F-box
/FBD/LRR-repeat protein, leucine-rich repeat receptor-like, Triose
phosphate isomerase cytosolic isoform-like protein, and UDP-
glycosyltransferase 87A1-like. On the other hand, GSE71561
aimed to elucidate the molecular regulation of peach fruit de-

velopment, particularly the interactions between the seed and
mesocarp tissues. In this dataset, isoleucine N-monooxygenase
2, myb-related protein, and transcription factor bHLH75-like
were among the most important genes identified. Enrichment
analysis was conducted on both datasets to gain insights into the
functional significance of the identified genes. In GSE71460,
the enrichment analysis revealed several enriched terms and pro-
cesses that suggested the involvement of these genes in various
cellular processes, metabolic activities, and molecular interac-
tions related to peach development.

In a previous study, the expression patterns of Arabidopsis-
SKP1-like (ASK) genes, which play important roles in ubiquitin-
mediated proteolysis and various biological processes, were ex-
amined [45]. The findings of that study shed light on the ex-
pression profiles of SKP1-like genes in plants. Our study, focus-
ing on gene expression during fruit development, shares similar-
ities with the previous study. Therefore, our results contribute
to understanding the roles of SKP1-like genes in peach devel-
opment. Another investigation explored the molecular mecha-
nisms of self-incompatibility in plants. It identified a Skp1-like
protein as a crucial component of a protein complex involved
in pollen compatibility [46]. Although the study’s focus was
on self-incompatibility, the importance of Skp1-like proteins in
plant reproductive processes can provide insights into peach fruit
development. Additionally, we observed differential expression
of triose phosphate isomerase, an essential enzyme in the glyox-
alase pathway, during peach development. This finding aligns
with a separate study that discussed the implications of triose
phosphate isomerase and the glyoxalase pathway in plant re-
sponses to abiotic stress and signaling [47]. While the latter
study primarily examined stress responses, their insights into
triose phosphate isomerase activity are valuable for understand-
ing its involvement in peach development. By comparing our
findings with the mentioned studies, we enhance the current un-
derstanding of gene expression patterns during peach develop-
ment and establish potential connections to processes such as
ubiquitin-mediated proteolysis, self-incompatibility, and stress
responses.

In the case of GSE71561, the enrichment analysis highlighted
several important gene-enriched pathways. These included cel-
lular anatomical entity, intracellular, catalytic activity, membrane-
bounded organelle, intracellular membrane-bounded organelle,
metabolic process, cellular process, response to stimulus, or-
ganelle, intracellular organelle, chloroplast, cytoplasm, plastid,
binding, thylakoid, transit peptide, chloroplast thylakoid, response
to stress, cellular metabolic process, membrane, developmen-
tal process, biosynthesis of secondary metabolites, oxidation-
reduction process, thylakoid, and stromule, metabolic pathways,
photosystem, thylakoid, plastid envelope, chloroplast envelope,
oxidoreductase activity, response to abiotic stimulus, anatomi-
cal structure development, thylakoid membrane, and chloroplast
thylakoid membrane. These enriched pathways provide insights
into the molecular processes and organelle-specific functions as-

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Rizk, 2023 Identifying and characterizing key gene families influencing Prunus persica plant development

Figure 3. Protein-protein interaction networks of the identified sequences from the MADS-box gene family (A), Homeobox transcription factor family (B), NAC

transcription factor family (C), and Receptor kinase-like gene family (D) visualized using the STRING database. The networks provide a comprehensive overview

of the protein interactions within each gene family, shedding light on potential functional associations and regulatory relationships among the identified proteins.

sociated with peach fruit development, particularly in relation
to chloroplast function, photosynthesis, and response to stress.
This findings from the enrichment analysis of GSE71561 align
with the study conducted by Chen et al. [48], which investi-
gated the regulation of chloroplast development in peach. The
enrichment analysis highlighted gene-enriched pathways associ-
ated with cellular anatomical entity, intracellular processes, cat-
alytic activity, membrane-bounded organelles, metabolic processes,
and response to stimulus, among others. These enriched path-
ways provide insights into the molecular processes and organelle-
specific functions involved in peach fruit development, particu-
larly in relation to chloroplast function, photosynthesis, and re-
sponse to stress.

The gene expression analysis contucted in ths study focused
on identifying genes associated with peach development in four
gene families. The results of our analysis revealed specific genes
within each family that contribute to peach development. These

findings are consistent with previous studies conducted in re-
lated species, providing valuable insights into the molecular mech-
anisms underlying peach development. In the Homeobox tran-
scription factor family, we identified 8 genes associated with
peach development. Previous research by Testone et al. [49]
highlighted the significance of homeodomain protein Kn1 and
homeobox-leucine zipper protein PROTODERMAL FACTOR 2
-like in this family, specifically in stem development and regulat-
ing elongation and lignification during primary growth. These
findings support our identification of Homeobox transcription
factor genes associated with peach development. Similarly, within
the Mad box gene family, we found 8 genes associated with
peach development. In a study by Wuddineh et al. [50], a signif-
icant gene, SEEDSTICK-like protein, was identified within this
family in switchgrass, a related plant species. Although their
research focused on genetically engineering switchgrass for bio-
fuel production, the presence of SEEDSTICK-like protein in

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Rizk, 2023 Identifying and characterizing key gene families influencing Prunus persica plant development

Figure 4. Gene Expression Analysis in GSE71460 and GSE71561 Datasets. (A) Heatmap illustrating the expression profiles of key genes associated with posthar-

vest textural changes in peaches in the GSE71460 dataset, which explores the effects of gibberellic acid application and cold storage. (B) Heatmap displaying the

expression patterns of critical genes related to the target trait in the GSE71561 dataset, which investigates peach fruit development, including interactions between

seed and mesocarp tissues. (C) Visualization of the protein-protein interaction (PPI) network for the genes identified in the GSE71460 dataset, revealing their

interactions. (D) Representation of the PPI network for the genes identified in the GSE71561 dataset, uncovering interplay among the identified genes. (E) Iden-

tification of enriched biological pathways associated with postharvest textural changes in peaches in the GSE71460 dataset, offering insights into the underlying

molecular mechanisms. (F) Discovery of enriched biological pathways linked to the investigated trait in the GSE71561 dataset, providing valuable insights into

fundamental molecular processes governing postharvest textural changes in peaches.

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Rizk, 2023 Identifying and characterizing key gene families influencing Prunus persica plant development

our analysis suggests its potential involvement in peach develop-
ment. Within the NAC gene family, we discovered 2 genes asso-
ciated with peach development, although their specific functions
in relation to peach development are unknown. While no direct
studies on the role of these specific genes in peach were found,
further investigation of NAC genes in other plant species may
provide insights into their functions in peach development. In
the receptor-kinase-like gene family, we identified a total of 80
genes associated with peach development. Noteworthy genes in
this family, such as LRR receptor-like serine/threonine-protein
kinase RPK2 and serine/threonine-protein kinase-like protein AC-
R4, have been reported in various studies across different plant
species. Although no specific studies have linked these genes
to peach development, their presence in our analysis suggests
their potential involvement in various peach developmental pro-
cesses.

Conclusion
In conclusion, this study employed two methods, gene identi-

fication and sequence alignment, and gene expression data analy-
sis, to investigate gene families related to Prunus persica (peach)
and gain insights into their evolutionary relationships and regu-
latory mechanisms.

The gene identification and sequence alignment method in-
volved analyzing gene families such as Homeobox, MADS-box,
NAC, and Receptor kinase-like. Gene identification was con-
ducted using local BLASTp analysis, and multiple sequence align-
ment was performed to assess sequence variation and construct
phylogenetic trees. The results provided valuable information
about gene counts, sequence lengths, and sequence variations
within each gene family, shedding light on their potential roles
and functions in the peach genome. The comparison with previ-
ous studies highlighted variations in gene counts and sequence
lengths among different species or datasets, suggesting differ-
ences in gene composition and abundance. The gene expres-
sion data analysis method focused on analyzing two datasets,
GSE71561 and GSE71460, using the GEO2R online tool. The
analysis provided insights into gene expression patterns and reg-
ulatory mechanisms underlying peach fruit development, ripen-
ing, and postharvest changes. Gene enrichment analysis revealed
differentially expressed genes and identified biological processes
and molecular functions associated with peach fruit development
and response to external stimuli such as cold storage and gib-
berellic acid application.

The findings from both methods contribute to a better under-
standing of the molecular processes governing complex pheno-
types in peaches. The gene identification and sequence align-
ment method elucidated the diversity and potential functional di-
vergence within gene families, providing insights into their evo-
lutionary dynamics and functional specialization. The gene ex-
pression data analysis method revealed gene expression patterns
and regulatory mechanisms underlying important developmental
processes in peach.

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	Abstract
	Introduction
	Methods
	Gene identification and sequence alignment
	Gene expression data analysis

	Results and Discussion
	 Gene identification and sequence variationa
	Protein-protein interaction analysis
	Gene differential expression analysis

	Conclusion

