Postharvest Biology and Technology 193 (2022) 112045

Contents lists available at ScienceDirect

Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

Characterization of miRNA-mediated auxin signaling during banana (Musa

spp.) fruit ripening
Xiangjin Kong a, b, Jun Zeng a, Ze Yun a, Chunhua Hu c, Bao Yang a, b, Hongxia Qu a, b,
Yueming Jiang a, b, Hong Zhu a, *
a
Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement & Guangdong Provincial Key Laboratory of Applied Botany, South
China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
b
University of Chinese Academy of Sciences, Beijing 100049, China
c
Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China

A R T I C L E I N F O A B S T R A C T

Keywords: Auxin signaling is essential for the whole plant life cycle and the core components of auxin signaling include the
Banana auxin receptor TIR/AFB, corepressor Aux/IAA, and transcription factor ARF. They jointly modulate a variety of
Ripening biological processes through regulating the expression of auxin-responsive genes. In the past decades, microRNAs
Auxin signaling
(miRNAs) have been well documented as vital regulators of gene expression. A group of miRNA families have
MiRNA
been found to target genes associated with auxin signal transduction, such as TIR/AFBs and ARFs. Banana is a
typical climacteric fruit that undergo dramatic physiological and biochemical changes during ripening process.
However, the role of auxin itself and miRNAs in auxin signaling during fruit ripening remain largely unknown in
banana. In this study, we comprehensively characterized three key gene families of auxin signaling pathways in
banana. There are 15 TIR/AFB, 50 Aux/IAA, and 49 ARF genes in the banana genome. Also, we identified
multiple miRNAs that can target members of auxin-related gene families, and the targeting relationship and
inhibitory effect of miR393, miR160 and miR167 on specific TIR/AFB and ARF family members were further
experimentally validated. In addition, the expression analysis of banana pulp after ethylene treatment revealed
the specific response of these auxin-related miRNAs to ethylene. Finally, a mode of action of miRNA-mediated
auxin signaling in ethylene-induced banana fruit ripening was proposed. Our results not only unraveled the
regulation of miRNA on banana auxin response, but also provided a reference for the study of miRNA-mediated
auxin signaling in the ripening of climacteric fruit.

1. Introduction
Auxin is the most important signaling molecule in plants with a
central role in numerous aspects of plant development, including
embryogenesis, apical dominance, flowering and fruit development
(Fleming, 2006). Remarkable breakthroughs have been made in un­
derstanding the biosynthesis, transport, and perception of auxin (Weij­
ers and Wagner, 2016). There are three key components of the auxin
signaling pathway: transport inhibitor resistant/auxin signaling F-box
(TIR/AFB) proteins, auxin/indole-3-acetic acid (Aux/IAA) transcrip­
tional corepressors, and auxin response factors (ARFs). TIR/AFB pro­
teins contain an F-box domain for tethering to other subunits in the SCF
complex and a leucine-rich repeat (LRR)-containing domain for the
interaction with auxin and Aux/IAA family genes (Parry et al., 2009).
Aux/IAA proteins interact with ARFs through shared C-terminal do­
mains and are recruited to the genome regions. At the N-terminal, they
generally have EAR/EAR-like repressor motifs and an area required for
TIR/AFB interaction (Guilfoyle, 2015). The ARF family of transcrip­
tional regulators with sequence specificity play a vital role in the tran­
scriptional response to auxin, where they can either activate or repress
the expression of auxin response genes by binding to the auxin response
element (AuxRE) site in their promoter regions (Tiwari et al., 2003). A
typical ARF is characterized by a highly conserved N-terminal B3-type
DNA binding domain (DBD) for the AuxRE motif recognition, a middle
region for transcriptional regulation, and a C-terminal dimerization
domain that interacts with Aux/IAAs (Piya et al., 2014). Gene families
associated with the auxin signaling pathway have been reported in some
model plants or crops. For example, there are seven TIR/AFB, 29

* Corresponding author.
E-mail address: zhuhong@scbg.ac.cn (H. Zhu).

https://doi.org/10.1016/j.postharvbio.2022.112045
Received 21 April 2022; Received in revised form 20 July 2022; Accepted 22 July 2022
Available online 28 July 2022
0925-5214/© 2022 Elsevier B.V. All rights reserved.
X. Kong et al.
Aux/IAA and 23 ARF genes in Arabidopsis genome (Parry et al., 2009),
while the rice genome encodes six TIR/AFB, 31 Aux/IAA and 25 ARF
genes (Wang et al., 2007).
Over the past few decades, microRNAs (miRNAs) have emerged as
critical regulators in diverse aspects of plant growth and development.
This major class of plant small RNAs are primarily engaged in post-
transcriptional regulation by either transcript cleavage or translational
repression of their target mRNAs (Axtell and Bartel, 2005). Among the
plant hormone response pathways, the relationship between auxin and
miRNA has been most studied. In Arabidopsis, 23 ARF transcription
factor family members have been identified, with at least five ARF genes
having miRNA complementary sites (Bonnet et al., 2010). Experiments
in Arabidopsis have confirmed that miR167 affects the development of
pistil, stamen and petal by regulating the expression of ARF6 and ARF8
(Wu et al., 2006), and miR160 governs the formation of root cap cells by
regulating the expression of ARF10 and ARF16 (Wang et al., 2005).
Upon eliminating the inhibition of miR160 on its target genes ARF16
and ARF17, the morphology of Arabidopsis can be altered, including
dwarf seedling, shortened roots, deformed and more cotyledons, and
delayed flowering (Mallory et al., 2005). In addition, miR390 mediates
the cleavage of the TAS3 gene and the generation of tasiRNAs that act on
ARF2, ARF3 and ARF4 to regulate lateral root development in Arabi­
dopsis (Yoon et al., 2009). In tomato, the down-regulation of ARF6 and
ARF8 by miR167 impairs floral development and female fertility (Liu
et al., 2014, 2014). Similarly, down-regulation of miR160 causes
increased expression of its targets ARF10/16/17, which regulates
auxin-mediated ovary patterning and flower abscission (Damodharan
et al., 2016). The involvement of miRNAs in auxin response may be an
ancient mechanism since both miR160-ARF and miR390-TAS3-ARF
pathways have also been reported in the ancient terrestrial bryo­
phytes, suggesting that this mechanism emerged early in the evolution
of higher plants (Axtell and Bartel, 2005). In addition to ARFs, auxin
receptors are also regulated by miRNA. In Arabidopsis, miR393-targeted
TIR1 and AFB1/2/3 encode F-box proteins, which play an essential role
in auxin signal transduction (Jones-Rhoades and Bartel, 2004). Over­
expression of miR393 in Arabidopsis inhibits auxin signal transduction
and improves plant resistance to bacterial infection (Navarro et al.,
2006). In rice, overexpressing miR393 can down-regulate the expression
of TIR/AFB family homologous genes, resulting in phenotype with
hallmarks of altered auxin signaling such as more tillers, enlarged leaf
inclination and altered root growth (Bian et al., 2012). Conceivably, the
miR393-TIR module is also highly conserved in plants, as it has been
reported in many plant species (Bai et al., 2017; Cai et al., 2017; Zhu
et al., 2019).
To study the regulatory relationship between hormones and miRNA,
exogenous hormones can be applied during plant growth and develop­
ment to analyze the expression changes of miRNA. Ethylene plays an
important role in plant life cycle, but there have been few reports on the
interaction between ethylene and miRNA (Liu and Chen, 2009). In rice,
both miR159 and miR394 are found to be regulated by ethylene (Liu
et al., 2009). In another study, roots of M. truncatula treated with
ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) show
altered accumulation of miRNAs such as miR159, miR164 and miR319
by high-throughput sequencing (Chen et al., 2012). These studies sug­
gest that some biological functions of ethylene may also depend on
miRNA pathways. However, few targets involving ethylene signal
transduction have been confirmed to be directly regulated by miRNAs
(Wang et al., 2018).
Banana (Musa spp.), an important economic fruit cultivated in South
China, usually undergo uncontrollably fast ripening once it is initiated,
leading to quality deterioration and commercial value loss. The ethylene
effect has been focused and its signal transduction pathway has been
well documented in fruit ripening (Inaba et al., 2007). However, fewer
studies demonstrate the role of auxin signaling in fruit ripening, and
whether and which key components of the auxin signaling pathway are
involved in banana fruit ripening remains largely elusive. Moreover, to
2
Postharvest Biology and Technology 193 (2022) 112045
what extent is the miRNA-TIR/ARF module conserved in banana is un­
clear. Therefore, we here comprehensively characterized these three key
gene families in auxin signaling in banana, where 15 TIR/AFB, 50
Aux/IAA and 49 ARF genes were identified. Gene structure, phylogeny,
and targeting relationship with miRNAs were also analyzed. The
expression patterns of TIRs, Aux/IAAs, and ARFs were examined during
ethylene-induced fruit ripening. Among them, miR393-TIR7, miR160-­
ARF12/18, and miR167-ARF7/35 modules were found to be mainly
associated with banana fruit ripening. Our results provided new
knowledge to study the function of plant miRNA-TIR/ARF modules in
fruit ripening.
2. Materials and methods
2.1. Plant materials and hormone treatment
Mature green banana fruit (‘Baxi’ at 110 d after anthesis) were
harvested from Wanqingsha plantain located at Panyu district in
Guangdong province. Uniform fruit fingers without defects were
selected and rinsed in 0.1% (w/v) Sporgon solution (Bayer, Germany)
for 3 min to minimize the potential microorganism effect. After air
drying, 180 fruit fingers were randomly selected and divided into two
groups: control (untreated) and exogenous hormone treatment. Each
group had three biological replicates with 30 fruit fingers in each
replicate. For ethylene treatment, the fingers were fumigated in 100 μL
L-1 ethylene for 18 h. Both fruit groups were packed with 0.02 mm-thick
polyethene films and stored at 25 ◦ C with 85–90% relative humidity.
Pulp tissues were taken from the middle of three fingers on 2, 4, 6, and 8
d, respectively, and immediately frozen with liquid nitrogen, ground
into small particles, and stored at − 80 ◦ C for later use. The sample on
0 d was taken as the starting point and shared by the control group and
the treatment group. Auxin treatment was performed with 0.1 mM
indole-3-acetic acid (IAA) solution for 10 min, and treated fruit were
stored according to the above-mentioned conditions.
2.2. Determination of physiological indicators related to banana fruit
ripening
Fruit ripening was evaluated by fruit peel color, firmness, respiratory
rate and ethylene release. In brief, peel color was determined using a
colorimeter CR-400 (Minolta Camera Co. Ltd., Japan). The penetrom­
eter GY-1 (Hangzhou Scientific Instruments, China) was used to measure
fruit firmness. The respiration rate was determined according to the
method of (Wang et al., 2013), and the ethylene production rate was
measured as described in (Pan et al., 2015). In addition, the IAA level
was determined during fruit ripening using the method in (Pan et al.,
2015) with modifications.
2.3. Identification of TIR/AFB, Aux/IAA, and ARF gene families in
banana
Complete protein sequences of banana (Musa acuminata) were
downloaded from the banana genome database (version 4.3) (http://
banana-genome.cirad.fr/) (Belser et al., 2021). Arabidopsis and rice
TIR/AFB, Aux/IAA, and ARF protein sequences were respectively ob­
tained from the TAIR (http://www.arabidopsis.org/) and the RGAP
(http://rice.plantbiology.msu.edu/) databases. Hidden Markov Model
(HMM) files corresponding to the TIR/AFB, Aux/IAA, and ARF domains
(PF18791, PF02309 and PF06507) were downloaded from PFAM pro­
tein family database (http://pfam.sanger.ac.uk/). HMMER 3.2
(http://www.hmmer.org/) was used to search the TIR/AFB, Aux/IAA,
and ARF genes from the banana genome. The default parameters were
used and the cutoff value was set to 0.01. Then, all TIR/AFB, Aux/IAA
and ARF protein sequences from Arabidopsis and rice were used as
reference sequences, and the potential TIR/AFBs, Aux/IAAs and ARFs in
banana database were further characterized by BLAST analysis using
X. Kong et al.
TBtools (Chen et al., 2020), with the cutoff e-value set at 1e-5. All
identified TIR/AFBs, Aux/IAAs, and ARFs in banana were further vali­
dated by conserved domain search using the PFAM and NCBI CDD
(http://www.ncbi.nlm.nih.gov/cdd/) databases.
2.4. Gene structure and conserved domains of TIR/AFBs, Aux/IAAs, and
ARFs in banana
For each gene member of the three gene families, the introns and
exons were analyzed using TBtools. The Batch-CD search (http://www.
ncbi. nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) was used to define the
conserved domain with default parameters. Conserved motifs were
analyzed using the MEME-Suite program (http://meme-suite.org) to
compare the structural differences among members of each gene family.
All results were visualized by TBtools.
2.5. Analysis of physical and chemical properties of TIR/AFBs, Aux/
IAAs, and ARFs in banana
ExPASy (https://web.expasy.org/protparam/) was used to analyze
the physical and chemical properties of the identified proteins, including
sequence length, molecular weight (MW) and isoelectric point (PI). The
subcellular localizations of these proteins were predicted by WoLF
PSORT (https://wolfpsort.hgc.jp/).
2.6. Characterization and validation of TIR/AFBs, Aux/IAAs, and ARFs
targeted by miRNAs in banana
In banana, most of the TIR/AFB- and ARF-targeting miRNAs have
been identified in our previous study via degradome sequencing, where
multiple targets with a high confidence level (all fell into category 0 with
alignment scores no more than 4) were screened (Zhu et al., 2019). To
comprehensively explore the targeting relationship between miRNAs
and their auxin-related target genes, all potential target sites were
computationally analyzed by TAPIR, a web server for plant microRNA
target prediction (http://bioinformatics.psb.ugent.be/webtools/tapir)
(Bonnet et al., 2010), and miRBase information (http://www.mirbase.
org/). To in vivo validate the interactions between miRNAs and their
targets, we used Agrobacterium tumefaciens infiltration to transiently
co-express miRNAs and their targets in the leaves, as described previ­
ously (Liu et al., 2014).
2.7. RNA blot of TIR/ARF-targeting miRNAs in banana
Total RNA of the pulp tissue sample was extracted from control and
ethylene-treated fruit by the in-house hot boron method. Then 20 μg of
total RNA was separated on a 15% polyacrylamide gel and then trans­
ferred to Hybond™-NX membranes (GE Healthcare, USA). UV light was
applied to crosslink the RNA samples before blotting. Probes of DNA
oligonucleotides reverse-complementary to banana miRNAs (Table S1)
were labeled by biotin at 3’ end and synthesized by Sangon Biotech.
Membranes were hybridized overnight at 37 ◦ C and washed three times
using the stringent buffer containing 1 x SSC and 1% SDS. After washing,
membranes were blocked and chromogenically reacted using Chemilu­
minescent Nucleic Acid Detection Module (Thermo, USA). Then a cold-
light CCD imager (Invitrogen, USA) was used for membrane scanning
and imaging. Membranes were also probed with a DNA molecule com­
plementary to U6 as the loading control.
2.8. Expression profiling of auxin signaling related genes and validation of
miRNA-targeted genes in banana
RNA-seq data for banana fruit ripening was downloaded from the
National Center for Biotechnology Information (NCBI). Based on the
sequences of the identified MaTIR/AFBs, MaAux/IAAs and MaARFs, a
BLASTN search was conducted to find their gene counterparts in the
3
Postharvest Biology and Technology 193 (2022) 112045
datasets (Li et al., 2019; Lü et al., 2018; Yun et al., 2019), and gene
expression profiles were then visualized by heatmaps using TBtools
(Fig. S1). To validate the gene expression profiling data, quantitative
real-time PCR of selected MaTIR/AFB and MaARF genes was carried out,
with the same RNA samples used for RNA blot analysis. First-strand
cDNA synthesis and qRT-PCR analyses were performed as described
previously (Zhu et al., 2020). Gene-specific primers for the target
MaTIR/AFBs and MaARFs and reference gene ribosomal protein S2
(MaRPS2) were designed by the Primer BLAST of NCBI (http://blast.
ncbi.nlm.nih.gov/) (Table S1). The qRT-PCR reactions were normalized
with the Ct (cycle threshold) value of the reference gene. Formula 2-ΔΔCt
was used to calculate the relative expression levels of target genes. For
each tested gene, the analysis was repeated three times with triplicate
PCR reactions.
2.9. Statistical analysis
Data presented in this study are the mean values of three biological
replicates, and are expressed as mean ± standard error. The significance
of the difference among different samples was calculated using the
student’s t-test in statistics package for the Social Sciences version 7.5
(SPSS, Inc., USA).
3. Results
3.1. Banana ripening characteristics and the effect of exogenous ethylene
treatment
The normal ripening process of banana fruit usually takes place
within a month. Fruit appearance did not change obviously until 20–22
d after storage. In stark contrast, however, ethylene-treated fruit ripened
more quickly, as manifested by peel color turning and pulp softening as
early as two days after storage. Compared with the control, the ripening
process after ethylene treatment can be shortened to a week, depending
on the ethylene concentration (Fig. 1A). In accordance with the outer
appearance, fruit color hue angle and firmness both decreased more
quickly in the ethylene-treated fruit, demonstrating that the peel color
turned from green to yellow and the fruit became soft. Moreover,
ethylene release and fruit respiration were enhanced during ethylene-
induced fruit ripening (Fig. 1B). To learn how auxin evolved in this
process, we also measured the IAA content in the fruit pulp. There was
no obvious fluctuation of auxin in the control, where IAA level slightly
went up and then down during the first eight days, while fruit at this
stage had not yet started ripening. Somewhat unexpectedly, the IAA
level began to increase from 2 d in the ethylene-treated fruit, with the
level higher than that of the control on 4 and 6 d when the fruit turned to
be fully ripe (Fig. 1 C). To further investigate the effect of exogenous
auxin on ripening, mature green fruit were treated with 0.1 mM IAA for
10 min. The result showed that IAA treatment could accelerate fruit
ripening, accompanied by earlier fruit yellowing and softening (Fig. 1D).
These findings stimulated our idea to systematically investigate the
auxin signal transduction pathway during banana fruit ripening, with
possible regulatory mechanisms from the perspective of auxin-related
gene family and miRNA regulation as well.
3.2. Characterization of key gene families for auxin signaling in banana
Firstly, we characterized the three key gene families of the classic
auxin signal transduction pathway. To identify all the related gene
family members in banana, both HMM search and BLAST analyses were
performed to search against banana genome database (version 4.3).
After eliminating redundant results and further conserved domain
validation, 15 TIR/AFBs, 50 Aux/IAAs and 49 ARFs were obtained
(Table S2). They were renamed from MaTIR1 to MaTIR15, MaIAA1 to
MaIAA50 and MaARF1 to MaARF49 according to the chromosomal
location, respectively. The 15 MaTIRs were from Chr03, Chr05, Chr06,
X. Kong et al. Postharvest Biology and Technology 193 (2022) 112045

Fig. 1. Changes of phenotype and physiology during banana fruit ripening at 25 ◦ C. (A) Phenotypic variation among naturally-ripen and ethylene-induced banana
fruit. (B) Change of peel color, firmness, respiration rate and ethylene production during natural and ethylene-induced fruit ripening. (C) Change of auxin level in an
8-d period. Data were presented as the means ± standard errors (n = 3) and significant differences were indicated with asterisks (* p < 0.05, ** p < 0.01). (D)
Accelerated ripening of banana fruit after 0.1 mM IAA treatment. Photo was taken after 15 d of storage.

Chr07, Chr08, Chr09 and Chr10. The 50 MaIAAs were distributed on
almost all chromosomes except Chr01, while 49 MaARFs were distrib­
uted on all chromosomes (Table S2). To explore the phylogenetic rela­
tionship of MaTIR, MaIAA and MaARF proteins, we constructed
neighbor-joining (NJ) trees, based on the amino acid sequences of
TIR/AFB, Aux/IAA and ARF proteins from banana, Arabidopsis and rice.
As shown in Fig. S2A, 28 TIR/AFB proteins from banana, Arabidopsis and
rice were separated into three classes. Among them, Class I contained 15
TIR proteins, Class II included eight and the remaining five belonged to
Class III. MaTIR2 and MaTIR5 showed a closer phylogenetic relationship
with AtTIR1, AtAFB1 and OsTIR1, while MaTIR12, MaTIR6, MaTIR10
and MaTIR14 were grouped with AtCOL1. The 110 IAAs were divided

4
X. Kong et al. Postharvest Biology and Technology 193 (2022) 112045

into seven classes, with Class I containing the most proteins (27 MaIAAs,
15 AtIAAs, 15 OsIAAs) and Class VII containing only two (AtIAA29 and
MaIAA25) (Fig. S2B). In addition, 97 ARF proteins were grouped into
three classes. Class I was the biggest group and Class III only included
two OsARFs (OsARF13 and OsARF20) (Fig. S2C).
3.3. Analysis of physicochemical properties of TIR/AFBs, Aux/IAAs, and
ARFs in banana
Based on our in-house transcriptome sequencing data, we further
screened out those members with no or very low expression, resulting in
12 TIR/AFBs, 10 Aux/IAAs and 35 ARFs, respectively. These members
were used for further analysis of structure evolution, with their gene
structure (intron/exon number and positions) and functional domains
presented based on phylogenetic analysis. WoLF PSORT was applied for
protein subcellular localization prediction. The members of both TIR/
AFB and Aux/IAA families were divided into three groups (Fig. 2). For
the TIR/AFBs, all members contained the transport inhibitor response 1
protein domains and F-box domains, but only two members had LRR
domains. The full length of these banana TIR/AFB proteins ranged from
575 to 651 aa, and their relative molecular mass varied from 64.12 to
72.86 kDa, with PIs in the range of 5.85–7.44. Most TIR/AFB family
members were predicted to be localized in the cytoplasm, with only a
few in the chloroplast, endoplasmic reticulum and nucleus. A total of 15
distinct conserved motifs (motif1–15) were identified and all MaTIR
proteins contained motif1, 2, 4, 5, 6, 7, 9, 10, 11, 12 and 13. Interest­
ingly, motif locations exhibited class-specific patterns. For example,
motif3 and motif8 were missing among Class III protein members, while
motif14 and motif15 were located only in the members of Class III and
Class I, respectively (Fig. S3A). For the Aux/IAAs, all protein sequences
contained Aux/IAA domains, with the full length ranging from 181 to
780 aa. The relative molecular mass of these banana Aux/IAA proteins
varied from 20.32 to 85.05 kDa and the PIs were in the range of
4.95–9.04. Aux/IAAs might be localized in the nucleus, chloroplast and
cytoplasm. Motif analysis showed that all MaIAA proteins contained
motif1, and almost all proteins had motif2 and motif3 except MaIAA40.
Motif14 and motif15 only existed in Class III (MaIAA48) and Class IV
(MaIAA40) (Fig. S3B). Furthermore, it confirmed the classification of
the 35 banana ARFs into four groups. As shown in Fig. 2, All ARFs had
typical ARF protein structure, which is composed of a highly conserved
DBD in the N-terminal region with a plant-specific B3-type subdomain
and an auxin-resp subdomain. In addition, 25 ARFs had the AUX_IAA
dimerization subdomains, indicating that these ARFs can interact with
each other or Aux/IAA proteins. The full length of these banana ARF
proteins ranged from 584 to 1137 aa, and their relative molecular mass
varied from 63.15 to 126.75 kDa, with PIs from 5.39 to 8.54. Subcellular
localization prediction showed that almost all ARF proteins were
localized in the nuclear except MaARF2/3/13 (Table S3). All MaARF
proteins conservatively contained motif1, 3, 4, 5, 6, 7, 10 and 11.
Specially, Class I and Class II possessed motif2 and Class I-III had
motif13. Motif14 was found in all members of Class IV but not other
classes, suggesting that it may be unique to Class IV (Fig. S3C). All these
results provided additional evidence confirming the phylogenic re­
lationships among members of the three key gene families for auxin
signaling in banana.
3.4. Analysis of miRNAs targeting auxin signaling-related genes in
banana
Combined with bioinformatic prediction and degradome data vali­
dation, multiple gene members of TIR/AFB and ARF families were found
to be the targets of miRNAs (Fig. 3). Notably, all the miRNA target sites
were located in the 3’ end of the target genes. More interestingly, the
targeting of miRNAs on these target genes had a distinct bias. For
example, miR393 specifically targeted members of the Class I TIR
family. It is known that AtTIR1, AtAFB1, AtAFB2, AtAFB3, OsTIR1 and
OsAFB2 are targets of miR393 (Navarro et al., 2006). As shown in
Fig. S2A, these six genes were all in the Class I TIR family, indicating that
miR393 may tend to target all the members of the Class I TIR family, but
not Class II or Class III. Similarly, miR160 and miR167 tended to target
only members of Class IV and a subgroup of Class I ARF family. In
addition, all of the miR167 target sites on ARF Class I subgroup members
were located at the C-terminal domain that interacts with Aux/IAA
proteins. However, most ARF Class IV members lacked this domain, and
hence tended to be targeted only by miR160.

Fig. 2. Auxin signaling related gene families in banana. In the center, a domain architecture of three core components (TIR/AFB, Aux/IAA and ARF) of auxin
responses in plants was presented. Around it is the gene family analysis for these three components in banana. For each gene family, a phylogenetic tree was
presented on the left, using the neighbor-joining method by ClustalX 2.0 and MEGA-X. The tree reliability was assessed using 2000 bootstrap replicates. Gene clusters
I, II, III and IV were indicated with different color blocks. On the right, the exon-intron structure and conserved domains of each family were visualized by TBtools,
with the information retrieved from banana genome (v4.3) and the PFAM and NCBI CDD search. Lengths of exons, introns and domains of each gene were exhibited
proportionally. Gene members with no expression based on previous transcriptome sequencing data are excluded from this figure.

5
X. Kong et al.
Fig. 3. Auxin signaling related gene families targeted by miRNAs in banana. For each gene family, the targeting of specific miRNAs on (A) TIR/AFBs and (B) ARFs
was presented either computationally predicted or validated by degradome analysis.
3.5. Validation of miRNA effect on specific auxin signaling-related genes
To further validate the effect of miRNAs on the specific auxin
signaling-related genes, we profiled the cleavage of three selected
miRNAs on their target genes with t-plots generated from degradome-
seq data, showing the abundance of cleaved tags relative to their posi­
tions in the transcripts (Fig. 4A). These miRNAs included miR393,
miR160 and miR167, targeting TIR7, ARF12 and ARF7, respectively
(Fig. 3). For each target gene, a clear cleavage was detected at the target
site, and all three targets belonged to category 0, where the only highest
peak corresponded with the cleavage site (Axtell and Meyers, 2018),
indicating that they were highly confident targets. Furthermore,
6
Postharvest Biology and Technology 193 (2022) 112045
transient expression assays in tobacco leaves were conducted to verify
the inhibitory effect of these miRNAs on their targets (Fig. 4B). In the
experiment, in addition to a miRNA overexpression vector and a GFP
vector carrying the target site, an empty vector that could not recognize
the target site and a GFP vector carrying a modified target site were set
as a negative control. The above four vectors were combined in pairs and
introduced in Agrobacterium. Three days after infiltrating into the to­
bacco leaves, it was observed that the green fluorescence signal was
weakened on the leaves where miRNA and its target site were
co-expressed, while the fluorescence signal for other combinations
remained unchanged (Fig. 4B). These results proved that the tested
miRNAs interacted with and suppressed their targets, thus inhibiting the
X. Kong et al. Postharvest Biology and Technology 193 (2022) 112045

Fig. 4. Target validation of miR393, miR160 and miR167 in banana. (A) Target plots (t-plots) from degradome analysis showing the cleavage of specific miRNAs on
TIR/AFBs and ARFs. The red dot indicates signatures consistent with miRNA-directed cleavage. (B) In vivo validation of specific miRNAs on TIR/AFBs and ARFs. Four
overexpression vectors were constructed for the transient expression assay in tobacco. Co-infiltrated leaves and control leaves were photographed at the 3rd day after
infiltration under bright light and UV light. Combinations of vectors used in the assay were shown on the leaves.

expression of GFP gene and leading to the weakening of green fluores­
cence signals. Therefore, the targeting relationship of miR393-TIR7,
miR160-ARF12 and miR167-ARF7 were confirmed in banana. As
mentioned above, there were more TIR/AFB and ARF genes identified as
targeted by miR393 and miR160/167, respectively with the target plots
provided in Fig. S4.
3.6. Expression analysis of miRNA-target pairs in banana during ripening
To explore the relationship between miRNA expression and
ethylene-induced banana ripening, expression analysis was performed
for certain miRNA-target pairs, including the above-mentioned three
pairs, in natural ripening and ethylene-induced ripening of fruit. The
miRNA accumulation levels in the pulp tissue were determined by RNA
gel blot, and expression differences were observed among these miR­
NAs, where the abundance of miR393 and miR167 was much higher
than that of miR160 in banana pulp (Fig. 5A). The expression of miR160
was hardly detected especially upon ethylene treatment. In general,
miR393 and miR167 displayed a similar pattern in expression. In the
control sample, they gradually decreased from 2 to 6 d but increased on
8 d. Interestingly, both miRNAs were suppressed by ethylene treatment
with a descending pattern in expression, where the inhibition of miR167
was more pronounced than that of miR393 (Fig. 5A). In parallel, we also
identified miR164 with a peculiar expression pattern, where the varia­
tion trend of miR164 and the three auxin-related miRNAs mentioned
above was consistent in the control fruit, but opposite in the ethylene-
treated fruit (Fig. 5A).
Next, the confirmed target genes were also tested for their expression
patterns during banana ripening. The expression of TIR7 targeted by
miR393 was increased in the ethylene-treated pulp tissue since 6 d and
remained high till 8 d (Fig. 5B). For the miR160-targeted ARF12 and
ARF18, both were increased after ethylene treatment, particularly on 6
d. Similarly, the expression level of ARF7 and ARF35 targeted by
miR167 maintained much higher in the ethylene-treated pulp compared
with the control until 8 d (Fig. 5C). Taken together, a good anti-
correlation between miRNA accumulation and their target expression

7
X. Kong et al. Postharvest Biology and Technology 193 (2022) 112045

Fig. 5. Expression analysis of miRNA-target pairs during banana fruit ripening. (A) Accumulation of miR393, miR160, miR167 and miR164 in banana pulp during
ripening. (B) Gene expression pattern of TIR/AFBs targeted by miR393 in banana pulp during ripening. (C) Gene expression pattern of ARFs targeted by miR160 and
miR167 in banana pulp during ripening. Data were presented as the means ± standard errors (n = 3) and significant differences were indicated with asterisks
(* p < 0.05, ** p < 0.01).

was observed, indicating that auxin signaling pathway has been
enhanced by ethylene treatment via suppressing the miRNA effect in the
banana pulp.
4. Discussion
Gene families associated with auxin signal transduction have been
characterized in various plant species, and multiple gene members play
important roles in diverse developmental and physiological processes
(Dharmasiri et al., 2005). However, fewer reports have been available

8
X. Kong et al.
for banana (Hu et al., 2015; Sun et al., 2019). In this study, a total of 15
TIR/AFBs, 50 Aux/IAA s and 49 ARFs were identified, using the latest
version of banana genome (Table S2), implying obvious gene family
expansion in banana compared to Arabidopsis and rice (Guo et al., 2021;
Salehin et al., 2015) (Fig. S2). Among them, only 12 TIR/AFBs, 10
Aux/IAAs and 35 ARFs were detected to be expressed during fruit
ripening, suggesting that some gene members, in particular Aux/IAAs,
might be pseudogenes or expressed in an inducible manner under
certain conditions. Moreover, the structural changes of these auxin
signaling pathway related gene family members partly reflected the
adaptation to the environment in the process of genome evolution
(Fig. 2).
During the ripening of banana fruit, various physiological and
biochemical changes occur, including chlorophyll breakdown, cell wall
degradation and aroma accumulation, thus forming the unique edible
quality. For decades, one of the research interests in banana has been
focused on ripening regulation, which is critical for improving its shelf
life and commercial export potential (Bapat et al., 2010). Ethylene has
been extensively studied (Klee and Giovannoni, 2011; Liu et al., 2015),
but much less is known about the auxin role in this process. One inter­
esting phenomenon found in our study was that auxin level in banana
fruit increased during ethylene-induced ripening, rather than decreasing
as previously reported in many fruit species (Böttcher et al., 2010; Buta
and Spaulding, 1994; Purgatto et al., 2002). This also prompted us to
explore the role of genes related to auxin signal transduction in fruit
ripening and how miRNA participated in controlling ripening by
mediating auxin signaling pathway. In grape, it has been reported that
the free IAA level declines before the onset of ripening with a substan­
tially increased level of IAA-amino acid conjugate (Böttcher et al.,
2010). Similar profiles of free IAA and IAA conjugates have also been
reported in tomato and strawberry (Given et al., 1988; Liu et al., 2005).
A recent study shows that auxin signaling declines at the onset of
ripening in the wild-type tomato fruit, and lower auxin levels positively
correlate with enhanced ethylene sensitivity (Shin et al., 2019). Auxin is
usually thought to inhibit fruit ripening because exogenous application
of auxin downregulates several major ripening-related cell wall enzymes
such as pectin methylesterase and polygalacturonase (Figueroa et al.,
2012; Villarreal et al., 2009). However, in climacteric peach and tomato
fruit, along with the production of climacteric ethylene, the IAA content
was greatly increased (Miller et al., 1987; Gillaspy et al., 1993). On the
other hand, IAA can enhance climacteric ethylene synthesis by inducing
the expression of a key enzyme, ACS (Abel and Theologis, 1996;
Bleecker and Kende, 2000). In our study, IAA level rose since 2 d, prior
to the color turning of banana fruit after ethylene treatment and
remained higher than that of the control, suggesting a positive corre­
lation with fruit ripening (Fig. 1 A and C). Similarly, a great number of
auxin signaling-related genes have been identified to be up-regulated
during banana fruit ripening and exogenous IAA treatment can accel­
erate ripening by enhancing the expression of many ripening-related
genes (Li et al., 2019; Yun et al., 2019). Such discrepancy in auxin
regulation of fruit ripening between banana and other reported fruit
may partly result from sampling and species specificity. On the other
hand, auxin concentration and treatment methods are also likely to
cause different results. Conceivably, there could be difference in the
auxin response between ethylene-induced and normal ripening, sug­
gesting that normally ripe banana and ethylene-enhanced ripe banana
may undergo different biological processes even though they both ripen
eventually.
Fruit ripening is under strict control of phytohormones. Ethylene and
ABA, the two most studied hormones, are thought to govern this process
in climacteric and non-climacteric fruit, respectively (Cherian et al.,
2014). Other hormones, including auxin, gibberellin, jasmonate and
brassinosteroid, have also been implicated in fruit ripening (Csukasi
et al., 2011; Kondo et al., 2007; Trainotti et al., 2007; Vardhini and Rao,
2002). Hormones do not regulate ripening alone but rather act in con­
cert with each other, and the interactions between multiple elements
9
Postharvest Biology and Technology 193 (2022) 112045
related to auxin and other hormone transduction pathways have been
reported. For example, in grape, ABA can increase the expression of GH3
that encodes IAA-amido synthetase for IAA-amino acid conjugation,
probably through binding to its promoter, leading to the decrease of IAA
level at the initiation of fruit ripening (Böttcher et al., 2010). It has been
hypothesized that the ABA: auxin ratio could be one of the signals that
trigger fruit ripening (Archbold and Dennis, 1984). Based on our results,
it could also be speculated that ethylene: auxin ratio may serve as an
indicator of banana ripening (Fig. 1B and C). Likewise, ethylene in­
terplays with auxin. It has been found that the expression of a great
number of auxin transcription factors like Aux/IAAs and ARFs are
regulated by both ethylene and auxin, implying a point of convergence
between the responses to the two hormones during tomato fruit ripening
(Jones et al., 2002). In peach, it has also been demonstrated that
ethylene and auxin can interact on each other’s signaling pathways
(Trainotti et al., 2007). More recently, the auxin-ethylene crosstalk
during ripening is extensively investigated in various fruit species such
as apple, papaya, and durian (Khaksar and Sirikantaramas, 2020; Yue
et al., 2020; Zhang et al., 2020).
With the deepening of miRNA research, it has been found that a
variety of miRNAs and hormone signals form regulatory networks to
jointly control plant growth and development (Liu and Chen, 2009).
Most studies focus on miRNA and hormone regulation in flower and root
development, while the interaction of various hormones in fruit
ripening, such as ethylene and auxin, needs to be further studied. Here,
our data confirmed that in banana, miR393, miR160 and miR167 tar­
geted specific members of the auxin signaling pathway gene families,
while ethylene treatment suppressed all three miRNAs, thus releasing
the inhibition of their targeted ARF genes and enhancing auxin signaling
during ethylene-induced fruit ripening. Also, it has been reported that
auxin signaling can be suppressed by miR164. One of the target genes of
miR164 is NAC1 which belongs to the NAM/ATAF/CUC gene family in
Arabidopsis. The role of NAC1 is to transmit auxin signal and promote
lateral root growth (Xie et al., 2002, 2000). It is found that miR164 can
degrade the NAC1 mRNA, thus down-regulating the auxin signal and
inhibiting lateral root growth (Guo et al., 2005). Meanwhile, auxin can
induce miR164 expression and promote NAC1 mRNA cleavage,
providing a homeostatic mechanism to downregulate auxin signals (Guo
et al., 2005). In our study, an obvious induction of miR164 by ethylene
treatment was observed, which was opposite to the expression patterns
of those auxin-related miRNAs, i.e., miR393, miR160 and miR167
(Fig. 5A). Our degradome data showed that miR164 conservatively
targeted members of the NAC gene family in banana. However, without
further analysis of the NAC target genes, it remains difficult to conclude
whether the miR164-NAC pathway plays a similar role in banana
ripening as in Arabidopsis root development. Based on the present
experimental results, we proposed a model to explain the role of auxin
signaling pathway in the ethylene-induced banana ripening (Fig. 6).
Upon ethylene treatment, fruit ripening was greatly induced and this
process was coupled to the increased auxin level and enhanced auxin
signaling, which was jointly mediated by the miR393-TIR7, miR160-­
ARF12/18 and miR167-ARF7/35 modules.
5. Conclusion
In this study, we characterized three key gene families of auxin
signaling and the regulation of miRNA on the auxin response in banana.
Meanwhile, expression profiles in the ethylene-treated fruit showed that
both miR393-TIR/AFB and miR160/167-ARF modules were actively
involved in banana fruit ripening, which likely played a predominant
role in this process. Combining with the changes in their expression
patterns, a mode of action of miRNA-mediated auxin signaling in
ethylene-induced banana fruit ripening was proposed. Our results
enriched the understanding of the association between miRNA and
hormone signaling responses, including ethylene and auxin, providing
ideas for further targeted regulation of key gene loci during fruit
X. Kong et al.
Fig. 6. Mode of miRNA-mediated auxin signaling during banana fruit ripening. Fruit ripening was induced by ethylene and this process was coupled to an elevated
level of auxin in fruit and enhanced auxin signaling, which was jointly mediated by the miR393-TIR7, miR160-ARF12/18 and miR167-ARF7/35 modules.
ripening.
CRediT authorship contribution statement
Hong Zhu designed and supervised the project. Xiangjin Kong and
Jun Zeng performed the experiments, analyzed data and prepared the
manuscript draft. Ze Yun and Bao Yang participated in the data analysis
and interpretation. Chunhua Hu, Hongxia Qu and Yueming Jiang helped
with the manuscript revision. All authors have approved the final
version of the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
I’ve shared the link to my data at Attach File step.
Acknowledgments
This work was supported by the Natural Science Foundation of
Guangdong Province (Nos. 2021A1515011258 and 2015A030313686)
and National Natural Science Foundation of China (No. 31772371).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.postharvbio.2022.112045.
10
Postharvest Biology and Technology 193 (2022) 112045
References
Abel, S., Theologis, A., 1996. Early genes and auxin action. Plant Physiol. 111, 9–17.
https://doi.org/10.1104/pp.111.1.9.
Archbold, D., Dennis, F., 1984. Quantification of free ABA and free and conjugated IAA
in strawberry achene and receptacle tissue during fruit development. J. Am. Soc.
Hortic. Sci. 109, 330–335.
Axtell, M.J., Bartel, D.P., 2005. Antiquity of microRNAs and their targets in land plants.
Plant Cell 17, 1658–1673. https://doi.org/10.1105/tpc.105.032185.
Axtell, M.J., Meyers, B.C., 2018. Revisiting criteria for plant microRNA annotation in the
era of big data. Plant Cell 30, 272–284. https://doi.org/10.1105/tpc.17.00851.
Bai, B., Bian, H., Zeng, Z., Hou, N., Shi, B., Wang, J., Zhu, M., Han, N., 2017. MiR393-
mediated auxin signaling regulation is involved in root elongation inhibition in
response to toxic aluminum stress in barley. Plant Cell Physiol. 58, 426–439. https://
doi.org/10.1093/pcp/pcw211.
Bapat, V.A., Trivedi, P.K., Ghosh, A., Sane, V.A., Ganapathi, T.R., Nath, P., 2010.
Ripening of fleshy fruit: molecular insight and the role of ethylene. Biotechnol. Ad.
28, 94–107. https://doi.org/10.1016/j.biotechadv.2009.10.002.
Belser, C., Baurens, F.C., Noel, B., Martin, G., Cruaud, C., Istace, B., Yahiaoui, N.,
Labadie, K., Hřibová, E., Doležel, J., Lemainque, A., Wincker, P., D’Hont, A., Aury, J.
M., 2021. Telomere-to-telomere gapless chromosomes of banana using nanopore
sequencing. Commun. Biol. 4. https://doi.org/10.1038/s42003-021-02559-3.
Bian, H., Xie, Y., Guo, F., Han, N., Ma, S., Zeng, Z., Wang, J., Yang, Y., Zhu, M., 2012.
Distinctive expression patterns and roles of the miRNA393/TIR1 homolog module in
regulating flag leaf inclination and primary and crown root growth in rice (Oryza
sativa). New Phytol. (196), 149–161. https://doi.org/10.1111/j.1469-
8137.2012.04248.x.
Bleecker, A.B., Kende, H., 2000. Ethylene: a gaseous signal moleculein plants. Annu Rev.
Cell Dev. Biol. 16, 1–18. https://doi.org/10.1146/annurev.cellbio.16.1.1.
Bonnet, E., He, Y., Billiau, K., van de Peer, Y., 2010. TAPIR, a web server for the
prediction of plant microRNA targets, including target mimics. Bioinformatics 26,
1566–1568. https://doi.org/10.1093/bioinformatics/btq233.
Böttcher, C., Keyzers, R.A., Boss, P.K., Davies, C., 2010. Sequestration of auxin by the
indole-3-acetic acid-amido synthetase GH3-1 in grape berry (Vitis vinifera L.) and the
proposed role of auxin conjugation during ripening. J. Exp. Bot. 61, 3615–3625.
https://doi.org/10.1093/jxb/erq174.
Buta, J.G., Spaulding, D.W., 1994. Changes in indole-3-acetic acid and abscisic acid
levels during tomato (Lycopersicon esculentum Mill.) fruit development and ripening.
J. Plant Growth Regul. 13, 163–166. https://doi.org/10.1007/BF00196382.
Cai, Z., Wang, Y., Zhu, L., Tian, Y., Chen, L., Sun, Z., Ullah, I., Li, X., 2017. GmTIR1/
GmAFB3-based auxin perception regulated by miR393 modulates soybean
nodulation. New Phytol. 215, 672–686. https://doi.org/10.1111/nph.14632.
Chen, C., Chen, H., Zhang, Y., Thomas, H.R., Frank, M.H., He, Y., Xia, R., 2020. TBtools:
an integrative toolkit developed for interactive analyses of big biological data. Mol.
Plant 13, 1194–1202. https://doi.org/10.1016/j.molp.2020.06.009.
X. Kong et al.
Chen, L., Wang, T., Zhao, M., Zhang, W., 2012. Ethylene-responsive miRNAs in roots of
Medicago truncatula identified by high-throughput sequencing at whole genome
level. Plant Sci. 184, 14–19. https://doi.org/10.1016/j.plantsci.2011.11.007.
Cherian, S., Figueroa, C.R., Nair, H., 2014. “Movers and shakers” in the regulation of fruit
ripening: a cross-dissection of climacteric versus non-climacteric fruit. J. Exp. Bot.
65, 4705–4722. https://doi.org/10.1093/jxb/eru280.
Csukasi, F., Osorio, S., Gutierrez, J.R., Kitamura, J., Giavalisco, P., Nakajima, M.,
Fernie, A.R., Rathjen, J.P., Botella, M.A., Valpuesta, V., Medina-Escobar, N., 2011.
Gibberellin biosynthesis and signalling during development of the strawberry
receptacle. New Phytol. 191, 376–390. https://doi.org/10.1111/j.1469-
8137.2011.03700.x.
Damodharan, S., Zhao, D., Arazi, T., 2016. A common miRNA160-based mechanism
regulates ovary patterning, floral organ abscission and lamina outgrowth in tomato.
Plant J. 86, 458–471. https://doi.org/10.1111/tpj.13127.
Dharmasiri, N., Dharmasiri, S., Weijers, D., Lechner, E., Yamada, M., Hobbie, L.,
Ehrismann, J.S., Jürgens, G., Estelle, M., 2005. Plant development is regulated by a
family of auxin receptor F box proteins. Dev. Cell 9, 109–119. https://doi.org/
10.1016/j.devcel.2005.05.014.
Figueroa, C.R., Opazo, M.C., Vera, P., Arriagada, O., Díaz, M., Moya-León, M.A., 2012.
Effect of postharvest treatment of calcium and auxin on cell wall composition and
expression of cell wall-modifying genes in the Chilean strawberry (Fragaria
chiloensis) fruit. Food Chem. 132, 2014–2022. https://doi.org/10.1016/j.
foodchem.2011.12.041.
Fleming, A.J., 2006. Plant signalling: the inexorable rise of auxin. Trends Cell Biol. 16,
397–402. https://doi.org/10.1016/j.tcb.2006.06.005.
Gillaspy, G., Ben-David, H., Gruissem, W., 1993. Fruits: a developmental perspective.
Plant Cell 5, 1439–1451. https://doi.org/10.1105/tpc.5.10.1439.
Given, N.K., Venis, M.A., Gierson, D., 1988. Hormonal regulation of ripening in the
strawberry, a non-climacteric fruit. Planta 174, 402–406. https://doi.org/10.1007/
BF00959527.
Guilfoyle, T.J., 2015. The PB1 domain in auxin response factor and Aux/IAA proteins: a
versatile protein interaction module in the auxin response. Plant Cell 27, 33–43.
https://doi.org/10.1105/tpc.114.132753.
Guo, F., Huang, Y., Qi, P., Lian, G., Hu, X., Han, N., Wang, J., Zhu, M., Qian, Q., Bian, H.,
2021. Functional analysis of auxin receptor OsTIR1/OsAFB family members in rice
grain yield, tillering, plant height, root system, germination, and auxinic herbicide
resistance. New Phytol. 229, 2676–2692. https://doi.org/10.1111/nph.17061.
Guo, H.S., Xie, Q., Fei, J.F., Chua, N.H., 2005. MicroRNA directs mRNA cleavage of the
transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root
development. Plant Cell 17, 1376–1386. https://doi.org/10.1105/tpc.105.030841.
Hu, W., Zuo, J., Hou, X., Yan, Y., Wei, Y., Liu, J., Li, M., Xu, B., Jin, Z., 2015. The auxin
response factor gene family in banana: genome-wide identification and expression
analyses during development, ripening, and abiotic stress. Front. Plant Sci. 6.
https://doi.org/10.3389/fpls.2015.00742.
Inaba, A., Liu, X., Yokotani, N., Yamane, M., Lu, W.J., Nakano, R., Kubo, Y., 2007.
Differential feedback regulation of ethylene biosynthesis in pulp and peel tissues of
banana fruit. J. Exp. Bot. 58, 1047–1057. https://doi.org/10.1093/jxb/erl265.
Jones, B., Frasse, P., Olmos, E., Zegzouti, H., Li, Z.G., Latché, A., Pech, J.C.,
Bouzayen, M., 2002. Down-regulation of DR12, an auxin-response-factor homolog,
in the tomato results in a pleiotropic phenotype including dark green and blotchy
ripening fruit. Plant J. 32, 603–613. https://doi.org/10.1046/j.1365-
313X.2002.01450.x.
Jones-Rhoades, M.W., Bartel, D.P., 2004. Computational identification of plant
microRNAs and their targets, including a stress-induced miRNA. Mol. Cell 14,
787–799. https://doi.org/10.1016/j.molcel.2004.05.027.
Khaksar, G., Sirikantaramas, S., 2020. Auxin response factor 2A is part of the regulatory
network mediating fruit ripening through auxin-ethylene crosstalk in durian. Front.
Plant Sci. 11. https://doi.org/10.3389/fpls.2020.543747.
Klee, H.J., Giovannoni, J.J., 2011. Genetics and control of tomato fruit ripening and
quality attributes. Annu. Rev. Genet. 45, 41–59. https://doi.org/10.1146/annurev-
genet-110410-132507.
Kondo, S., Yamada, H., Setha, S., 2007. Effect of jasmonates differed at fruit ripening
stages on 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase gene
expression in pears. J. Am. Soc. Hortic. Sci. 132, 120–125. https://doi.org/
10.21273/jashs.132.1.120.
Li, T., Yun, Z., Wu, Q., Qu, H., Duan, X., Jiang, Y., 2019. Combination of transcriptomic,
proteomic, and metabolomic analysis reveals the ripening mechanism of banana
pulp. Biomolecules 9. https://doi.org/10.3390/biom9100523.
Liu, K., Kang, B.C., Jiang, H., Moore, S.L., Li, H., Watkins, C.B., Setter, T.L., Jahn, M.M.,
2005. A GH3-like gene, CcGH3, isolated from Capsicum chinense L. fruit is regulated
by auxin and ethylene. Plant Mol. Biol. 58, 447–464. https://doi.org/10.1007/
s11103-005-6505-4.
Liu, M., Pirrello, J., Chervin, C., Roustan, J.P., Bouzayen, M., 2015. Ethylene control of
fruit ripening: revisiting the complex network of transcriptional regulation. Plant
Physiol. 169, 2380–2390. https://doi.org/10.1104/pp.15.01361.
Liu, N., Wu, S., Houten, J., van, Wang, Y., Ding, B., Fei, Z., Clarke, T.H., Reed, J.W., van
der Knaap, E., 2014. Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by
microRNA 167 leads to floral development defects and female sterility in tomato.
J. Exp. Bot. 65, 2507–2520. https://doi.org/10.1093/jxb/eru141.
Liu, Q., Chen, Y.Q., 2009. Insights into the mechanism of plant development: interactions
of miRNAs pathway with phytohormone response. Biochem. Bioph. Res. Co. 384,
1–5. https://doi.org/10.1016/j.bbrc.2009.04.028.
Liu, Q., Zhang, Y.C., Wang, C.Y., Luo, Y.C., Huang, Q.J., Chen, S.Y., Zhou, H., Qu, L.H.,
Chen, Y.Q., 2009. Expression analysis of phytohormone-regulated microRNAs in
rice, implying their regulation roles in plant hormone signaling. FEBS Lett. 583,
723–728. https://doi.org/10.1016/j.febslet.2009.01.020.
11
Postharvest Biology and Technology 193 (2022) 112045
Liu, Y., Wang, L., Chen, D., Wu, X., Huang, D., Chen, L., Li, L., Deng, X., Xu, Q., 2014.
Genome-wide comparison of microRNAs and their targeted transcripts among leaf,
flower and fruit of sweet orange. BMC Genom. 15. https://doi.org/10.1186/1471-
2164-15-695.
Lü, P., Yu, S., Zhu, N., Chen, Y.R., Zhou, B., Pan, Y., Tzeng, D., Fabi, J.P., Argyris, J.,
Garcia-Mas, J., Ye, N., Zhang, J., Grierson, D., Xiang, J., Fei, Z., Giovannoni, J.,
Zhong, S., 2018. Genome encode analyses reveal the basis of convergent evolution of
fleshy fruit ripening. Nat. Plants 4, 784–791. https://doi.org/10.1038/s41477-018-
0249-z.
Mallory, A.C., Bartel, D.P., Bartel, B., 2005. MicroRNA-directed regulation of Arabidopsis
auxin response factor17 is essential for proper development and modulates
expression of early auxin response genes. Plant Cell 17, 1360–1375. https://doi.org/
10.1105/tpc.105.031716.
Miller, A.N., Walsh, C.S., Cohen, J.D., 1987. Measurement of indole-3-acetic acid in
peach fruits (Prunus persica L. Batsch cv Redhaven) during development. Plant
Physiol. 84, 491–494. https://doi.org/10.1104/pp.84.2.491.
Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M., Voinnet, O.,
Jones, J.D.G., 2006. A plant miRNA contributes to antibacterial resistance by
repressing auxin signaling. Science 312, 436–439. https://doi.org/10.1126/
science.1126088.
Pan, L., Zeng, W., Niu, L., Lu, Z., Liu, H., Cui, G., Zhu, Y., Chu, J., Li, W., Fang, W., Cai, Z.,
Li, G., Wang, Z., 2015. PpYUC11, a strong candidate gene for the stony hard
phenotype in peach (Prunus persica L. Batsch), participates in IAA biosynthesis
during fruit ripening. J. Exp. Bot. 66, 7031–7044. https://doi.org/10.1093/jxb/
erv400.
Parry, G., Calderon-Villalobos, L.I., Prigge, M., Peret, B., Dharmasiri, S., Itoh, H.,
Lechner, E., Gray, W.M., Bennett, M., Estelle, M., 2009. Complex regulation of the
TIR1/AFB family of auxin receptors. P. Natl. Acad. Sci. USA (106,), 22540–22545.
https://doi.org/10.1073/pnas.0911967106.
Piya, S., Shrestha, S.K., Binder, B., Neal Stewart, C., Hewezi, T., 2014. Protein-protein
interaction and gene co-expression maps of ARFs and Aux/IAAs in Arabidopsis. Front.
Plant Sci. 5. https://doi.org/10.3389/fpls.2014.00744.
Purgatto, E., Oliveira Do Nascimento, J.R., Lajolo, F.M., Cordenunsi, B.R., 2002. The
onset of starch degradation during banana ripening is concomitant to changes in the
content of free and conjugated forms of indole-3-acetic acid. J. Plant Physiol. 159,
1105–1111. https://doi.org/10.1078/0176-1617-00875.
Salehin, M., Bagchi, R., Estelle, M., 2015. ScfTIR1/AFB-based auxin perception:
mechanism and role in plant growth and development. Plant Cell 27, 9–19. https://
doi.org/10.1105/tpc.114.133744.
Shin, J.H., Mila, I., Liu, M., Rodrigues, M.A., Vernoux, T., Pirrello, J., Bouzayen, M.,
2019. The RIN-regulated small auxin-up RNA SAUR69 is involved in the unripe-to-
ripe phase transition of tomato fruit via enhancement of the sensitivity to ethylene.
New Phytol. 222, 820–836. https://doi.org/10.1111/nph.15618.
Sun, X., Liu, F., Tian, N., Xiang, L., Hao, X., Wang, Y., Peng, L., Wang, T., Cheng, C.,
Lai, Z., 2019. Genome-wide identification and expression analysis of Aux/IAA gene
family in banana. Acta Hortic. Sin. 46, 1919–1935. https://doi.org/10.16420/j.
issn.0513-353x.2018-0743.
Tiwari, S.B., Hagen, G., Guilfoyle, T., 2003. The roles of auxin response factor domains in
auxin-responsive transcription. Plant Cell 15, 533–543. https://doi.org/10.1105/
tpc.008417.
Trainotti, L., Tadiello, A., Casadoro, G., 2007. The involvement of auxin in the ripening
of climacteric fruits comes of age: the hormone plays a role of its own and has an
intense interplay with ethylene in ripening peaches. J. Exp. Bot. 58, 3299–3308.
https://doi.org/10.1093/jxb/erm178.
Vardhini, B.V., Rao, S.S.R., 2002. Acceleration of ripening of tomato pericarp discs by
brassinosteroids. Phytochemistry 61, 843–847. https://doi.org/10.1016/S0031-
9422(02)00223-6.
Villarreal, N.M., Martínez, G.A., Civello, P.M., 2009. Influence of plant growth regulators
on polygalacturonase expression in strawberry fruit. Plant Sci. 176, 749–757.
https://doi.org/10.1016/j.plantsci.2009.02.019.
Wang, D., Pei, K., Fu, Y., Sun, Z., Li, S., Liu, H., Tang, K., Han, B., Tao, Y., 2007. Genome-
wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa).
Gene 394, 13–24. https://doi.org/10.1016/j.gene.2007.01.006.
Wang, H., Qian, Z., Ma, S., Zhou, Y., Patrick, J.W., Duan, X., Jiang, Y., Qu, H., 2013.
Energy status of ripening and postharvest senescent fruit of litchi (Litchi chinensis
Sonn.). BMC Plant Biol. 13. https://doi.org/10.1186/1471-2229-13-55.
Wang, J.W., Wang, L.J., Mao, Y.B., Cai, W.J., Xue, H.W., Chen, X.Y., 2005. Control of
root cap formation by microRNA-targeted auxin response factors in Arabidopsis.
Plant Cell 17, 2204–2216. https://doi.org/10.1105/tpc.105.033076.
Wang, Y., Zou, W., Xiao, Y., Cheng, L., Liu, Y., Gao, S., Shi, Z., Jiang, Y., Qi, M., Xu, T.,
Li, T., 2018. MicroRNA1917 targets CTR4 splice variants to regulate ethylene
responses in tomato. J. Exp. Bot. 69, 1011–1025. https://doi.org/10.1093/jxb/
erx469.
Weijers, D., Wagner, D., 2016. Transcriptional responses to the auxin hormone. Annu.
Rev. Plant Biol. 67, 539–574. https://doi.org/10.1146/annurev-arplant-043015-
112122.
Wu, M.F., Tian, Q., Reed, J.W., 2006. Arabidopsis microRNA167 controls patterns of
ARF6 and ARF8 expression, and regulates both female and male reproduction.
Development 133, 4211–4218. https://doi.org/10.1242/dev.02602.
Xie, Q., Frugis, G., Colgan, D., Chua, N.H., 2000. Arabidopsis NAC1 transduces auxin
signal downstream of TIR1 to promote lateral root development. Gene. Dev. 14,
3024–3036. https://doi.org/10.1101/gad.852200.
Xie, Q., Guo, H.S., Dallman, G., Fang, S., Weissman, A.M., Chua, N.H., 2002. SINAT5
promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals. Nature
419, 167–170. https://doi.org/10.1038/nature00998.
X. Kong et al.
Yoon, E.K., Yang, J.H., Lim, J., Kim, S.H., Kim, S.K., Lee, W.S., 2009. Auxin regulation of
the microRNA390-dependent transacting small interfering RNA pathway in
Arabidopsis lateral root development. Nucleic Acids Res 38, 1382–1391. https://doi.
org/10.1093/nar/gkp1128.
Yue, P., Lu, Q., Liu, Z., Lv, T., Li, X., Bu, H., Liu, W., Xu, Y., Yuan, H., Wang, A., 2020.
Auxin-activated MdARF5 induces the expression of ethylene biosynthetic genes to
initiate apple fruit ripening. New Phytol. 226, 1781–1795. https://doi.org/10.1111/
nph.16500.
Yun, Z., Li, T., Gao, H., Zhu, H., Gupta, V.K., Jiang, Y., Duan, X., 2019. Integrated
transcriptomic, proteomic, and metabolomics analysis reveals peel ripening of
harvested banana under natural condition. Biomolecules 9. https://doi.org/
10.3390/biom9050167.
12
Postharvest Biology and Technology 193 (2022) 112045
Zhang, T., Li, W., Xie, R., Xu, L., Zhou, Y., Li, H., Yuan, C., Zheng, X., Xiao, L., Liu, K.,
2020. CpARF2 and CpEIL1 interact to mediate auxin-ethylene interaction and
regulate fruit ripening in papaya. Plant J. 103, 1318–1337. https://doi.org/
10.1111/tpj.14803.
Zhu, H., Chen, C., Zeng, J., Yun, Z., Liu, Y., Qu, H., Jiang, Y., Duan, X., Xia, R., 2020.
MicroRNA528, a hub regulator modulating ROS homeostasis via targeting of a
diverse set of genes encoding copper-containing proteins in monocots. New Phytol.
225, 385–399. https://doi.org/10.1111/nph.16130.
Zhu, H., Zhang, Y., Tang, R., Qu, H., Duan, X., Jiang, Y., 2019. Banana sRNAome and
degradome identify microRNAs functioning in differential responses to temperature
stress. BMC Genom. 20. https://doi.org/10.1186/s12864-018-5395-1.