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Identification and evolution
analysis of YUCCA genes of
Medicago sativa and Medicago
truncatula and their expression
profiles under abiotic stress
An Shao, Shugao Fan, Xiao Xu, Wei Wang*and Jinmin Fu*
Coastal Salinity Tolerant Grass Engineering and Technology Research Center, Ludong University,
Yantai, Shandong, China
The YUCCAs (YUC) are functionally identified flavin-containing monooxidases
(FMOs) in plants that act as an important rate-limiting enzyme functioning in the
auxin synthesis IPA (indole-3-pyruvic acid) pathway. In this study, 12 MsYUCs and
15 MtYUCs containing characteristic conserved motifs were identified in M. sativa
(Medicago sativa L.) and M. truncatula (Medicago truncatula Gaertn.),
respectively. Phylogenetic analysis revealed that YUC proteins underwent an
evolutionary divergence. Both tandem and segmental duplication events were
presented in MsYUC and MtYUC genes. Comparative syntenic maps of M. sativa
with M. truncatula,Arabidopsis (Arabidopsis thaliana), or rice (Oryza sativa L.)
were constructed to illustrate the evolution relationship of the YUC gene family.
A large number of cis-acting elements related to stress response and hormone
regulation were revealed in the promoter sequences of MsYUCs. Expression
analysis showed that MsYUCs had a tissue-specific, genotype-differential
expression and a differential abiotic stress response pattern based on
transcriptome data analysis of M. sativa online. In addition, RT-qPCR confirmed
that salt stress significantly induced the expression of MsYUC1/MsYUC10 but
significantly inhibited MsYUC2/MsYUC3 expression and the expression of
MsYUC10/MsYUC11/MsYUC12 was significantly induced by cold treatment.
These results could provide valuable information for functional analysis of YUC
genes via gene engineering of the auxin synthetic IPA pathway in Medicago.
KEYWORDS
Medicago, YUC, evolution analysis, expression profile, abiotic stress response
1 Introduction
Auxin is a critical plant hormone, involved in diverse developmental events such as cell
division, cell differentiation, and flower development. Indole-3-acetic acid (IAA) is the
best-studied naturally occurring active auxin, which are synthesized by two pathways:
tryptophan-dependent pathway and tryptophan-independent pathway (Zhao, 2010). For
tryptophan-dependent IAA synthesis, there are four proposed branches: (1) indole-3-
Frontiers in Plant Science frontiersin.org01
OPEN ACCESS
EDITED BY
Hui Song,
Qingdao Agricultural University, China
REVIEWED BY
Xianqin Lu,
Shandong University, China
Yongzhe Ren,
Henan Agricultural University, China
*CORRESPONDENCE
Wei Wang
weiwang@ldu.edu.cn
Jinmin Fu
turfcn@qq.com
RECEIVED 27 July 2023
ACCEPTED 11 August 2023
PUBLISHED 28 August 2023
CITATION
Shao A, Fan S, Xu X, Wang W and Fu J
(2023) Identification and evolution analysis
of YUCCA genes of Medicago sativa and
Medicago truncatula and their expression
profiles under abiotic stress.
Front. Plant Sci. 14:1268027.
doi: 10.3389/fpls.2023.1268027
COPYRIGHT
© 2023 Shao, Fan, Xu, Wang and Fu. This is
an open-access article distributed under the
terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that
the original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms.
TYPE Original Research
PUBLISHED 28 August 2023
DOI 10.3389/fpls.2023.1268027
pyruvic acid (IPA); (2) tyramine pathway; (3) index-3-acetamide
pathway; and (4) index-3-acetoxime pathway (Stepanova et al.,
2011). Among the four branches, the IPA branch is the major route
of IAA biosynthesis inferred by the pleiotropic abnormal phenotype
of Arabidopsis mutants (Mashiguchi et al., 2011;Won et al., 2011).
In the initial step, IPA is catalyzed by Trp aminotransferase 1
(TAA1) and its related proteins TAR1 and TAR2 with Trp as the
precursor. Subsequently, the YUCCA (YUC)-encoded enzyme
catalyzes the generation of IAA by IPA (Fraaije et al., 2002;Won
et al., 2011). The YUC enzyme is the first functionally identified
flavin-containing monooxidase (FMOs) in plants. The conserved
domain of FMOs contains two conserved motifs, the flavin purine
dinucleotide (FAD) binding site and the reduced coenzyme binding
site (NADPH-binding site), which have the same G
X
GxxG
characteristic structure in their amino acid sequences (Zhao, 2012).
The YUC gene was originally identified from Arabidopsis
mutants with reduced IAA content (Zhao et al., 2001). Genetic
and physiological analyses of the loss-of-function mutants of the
YUC gene have further demonstrated its important role and rate-
limiting enzyme function in the auxin synthesis IPA pathway.
Overexpression of transgenic Arabidopsis lines of the YUC gene
showed slightly increased auxin levels, accompanied by phenotypic
including hypocotyl elongation, cotyledon bias, and enhanced
apical dominance (Zhao et al., 2001). Subsequent studies showed
that overexpression of the YUC gene in plants such as rice, potato,
and strawberry could also produce similar phenotypes of auxin
overproduction (Kim et al., 2012;Liu et al., 2014). In addition,
inactivation of a single YUC gene in Arabidopsis presented not
obvious developmental defects, whereas multiple mutants plants
have more severe phenotypes (Cheng et al., 2006), suggesting
functional redundancy among YUC members. Moreover, gene
and protein expression data in Arabidopsis indicated that YUC1,
2,4, and 6were mainly expressed in the stems, whereas YUC 3,5,7,
8, and 9were mainly functional in the roots (Chen et al., 2014). The
yuc1yuc2yuc4yuc6 quadruple mutants had severe defects in vascular
patterning and failed to produce a normal inflorescence but had no
root defects, consistent with their stem-localized expression pattern.
YUC3,5,7,8, and 9are expressed during root development, and the
multiple mutants of the five YUC genes developed short and
agrotropic roots (Chen et al., 2014). In addition, YUC genes
expressed in the shoots (YUC 1,2,4.1, and 6) are localized to the
cytoplasm, whereas root YUC genes are the ER (endoplasmic
reticulum) membrane-binding proteins. In addition, the
phenotypes of different sets of individual YUC knockout mutants
cannot be complemented by the expression of YUC genes expressed
in other tissues (Chen et al., 2014;Zhao, 2018). These studies
suggested that different sets of YUC genes exhibited tissue
expression specificity, organ-specific subcellular localization
patterns, and differential of gene function for auxin biosynthesis.
Plants often respond to environmental stress by regulating
hormonal pathways. Several studies have shown that the auxin
biosynthetic pathway is upregulated in response to certain abiotic
stresses including regulating the expression of YUC genes (Blakeslee
et al., 2019). For example, several root-specificYUC genes have been
reported to mediate aluminum stress-induced inhibition of root
growth in Arabidopsis (Liu et al., 2016). Heat and low-temperature
stress can induce ER sheet formation by inducing a specificYUC
gene (Pain et al., 2019). In Arabidopsis, heat stress led to an indirect
increased expression of YUC8 (Sun et al., 2012), which is similar to
the upregulation of CsYUC8/9 in cucumber. Cold stress also led to
the upregulation of CsYUC10b but downregulation of other CsYUC
proteins in cucumber (Yan et al., 2016). RNA-seq analysis of
Arabidopsis under heat and drought stress also revealed a tissue-
specific difference in the up- or downregulation of TAA/YUC auxin
biosynthesis genes, such as the upregulation of YUC9 expression in
leaf tissues after heat stress (Blakeslee et al., 2019). Overexpression
of YUC7 in Arabidopsis (Lee et al., 2012), and YUC6 in potato was
able to increase drought tolerance with reduced water loss in
transgenic plants by reducing the decomposition of IAA (Kim
et al., 2012;Cha et al., 2015). An increased free IAA level and
improved drought stress tolerance connected with reduced levels of
reactive oxygen species and delayed leaf senescence have been
observed for plants such as tomato, maize, rice, and petunia (Ke
et al., 2015). In contrast to most results in Arabidopsis, increased
drought tolerance associated with decreased root IAA levels in rice
was found, accompanied by the downregulation of various YUC
genes (Du et al., 2013;Naser and Shani, 2016). The different
expression patterns of YUC genes in response to different stresses
or in different species suggested a possible functional differentiation
of YUC genes during stress response.
Medicago sativa L. is a perennial herbaceous legume forages
with high yield, nutrient value, and palatability. As a basic
component in rations for animals and an important cash crop for
biofuel ethanol production, it is widely cultivated (Li et al., 2011).
However, the growth and yield of M. sativa could be severely
inhibited by external stresses such as salt, cold, and drought stress.
Recently, large-scale potential genes involved in M. sativa
responsive to adverse stimuli have been investigated by
transcriptional profiling and detected several stress-responsive
genes and categories (Postnikova et al., 2013;An et al., 2016;Luo
et al., 2019;Ma et al., 2021). Root and leaf transcriptomes under salt
stress revealed a hormone interaction involved in salinity
adaptation (Lei et al., 2018). Overexpressing IAA within root
nodules of M. sativa was associated with the improved drought
tolerance of plants (Defez et al., 2017). Although YUCs have been
identified in several species of plants, such as 11 AtYUCs in
Arabidopsis (Mashiguchi et al., 2011), 7 OsYUCs in rice
(Yamamoto et al., 2007), 22 TaYUCs in wheat (Yang et al., 2012),
22 GmYUCs in soybean (Wang et al., 2017) and 14 ZmYUCs in
maize (Li et al., 2015), the IAA biosynthesis-related YUC genes in
M. sativa or its model legume species M. truncatula (Medicago
truncatula Gaertn.) has not yet been identified at the genome-wide
level and the tissue-specific and abiotic stress expression patterns
have not been analyzed (Li and Brummer, 2012), greatly limiting
the improvement of stress adaptability of M. sativa by modifying
the auxin pathway through genetic engineering.
In this study, a total of 12 MsYUCs in M. sativa and 15 MtYUCs
in M. truncatula were identified. The gene structure, motif
composition, chromosome location, and gene replication events
were analyzed, and the evolutionary relationship of other species
associated with M. sativa was constructed. An overall comparative
expression analysis in M. sativa was performed to examine the YUC
Shao et al. 10.3389/fpls.2023.1268027
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gene expression patterns in different tissues, different varieties, and
their responses to cold, drought, and salt stress. These results could
provide valuable information for identifying candidate MsYUCs
involved in different biological processes and various abiotic stress
responses in M. sativa for further gene functional study and for
genetic modification.
2 Materials and methods
2.1 Identification of Medicago YUC genes
and basic characteristic analysis
Genome sequence and genome annotation information of
M. sativa variety Zhongmu No. 1 and M. truncatula used in this
study were downloaded from Ensembl Plants (https://
plants.ensembl.org). The amino acid sequences of the Arabidopsis
YUC family members were downloaded from the TAIR website
(https://www.arabidopsis.org/) and used as Query to search the
Medicago protein sequences by Local BLAST, and the sequences
with e-value less than −20 were reserved. The latest version of all
schema database files “Pfam-a.hm.gz”from the Pfam database
(https://pfam.xfam.org/) were downloaded, and candidate YUC
members containing the FMO-like domain (PFam00743)
were identified using TBtools’(Chen et al., 2020) simple HMM
Search plug-in. Results obtained from BLAST and Pfam search were
further merged to remove duplicates. Finally, the Batch CD search
function in the NCBI website (https://www.ncbi.nlm.nih.gov/) and
the SMART database were used to detect and retain the correct and
complete sequences of YUC characteristic conserved motifs (http://
smart.embl-heidelberg.de/). The basic features such as molecular
weight were determined, and isoelectric point analysis was
performed using the ExPASy Proteomic Server (https://
web.expasy.org/protparam/).
2.2 Chromosome localization and
conserved motif and gene
structure analyses
According to the chromosomal location data contained in
the downloaded Medicago genome annotation information,
TBtools was used to map the chromosomal location of YUC
members. The YUC members detected in Medicago were named
according to their position from top to bottom on chromosomes
1–8. The conserved motif of YUC genes was identified using
online motif detection software (http://meme.nbcr.net/meme/),
and the length of the motif was set from 2 to 200 bp to detect a
maximum of 12 motifs. Visualization was performed with the
TBtools software. For gene structure analysis, TBtools’“gene
structure view”function was used to visualize the gene structure
(exon and intron number and location) of MsYUC family genes.
The “One step build ML tree”plug-in of TBtools was used to get
a Newick tree and displayed in the front of the conversed motif
and gene structure exhibition.
2.3 Phylogenetic and gene duplication and
synteny analysis
A phylogenetic tree was constructed using the YUC amino acid
sequences of Arabidopsis, rice, M. truncatula, and M. sativa to
analyze the homology relationships. All YUC sequences were
aligned to multiple sequences using Clustal W, and the alignment
resulted in phylogenetic tree construction using MEGA6.0 software
(Larkin et al., 2007;Tamura et al., 2013). The establishment method
used the adjacency method (neighbor-joining method) and the P-
distance model with the bootstrap test for 1,000 times. Replication
events of Medicago YUC genes and collinear blocks of YUC genes
within M. sativa,Arabidopsis,M. truncatula, and rice were analyzed
using the “One Step MCScanX Wrapper”function of TBtools with
the e-value of 1e
−3
and number of blast hits of 10. Tandem and
segmental duplicates in the YUC gene family were identified using
TBtools by searching the final “tandem”and “gene Linked Region”
files after running. Phylogenetic analysis of species was performed
using “phyliptree.phy”derived from the “NCBI Taxonomy”
function. The Ka (nonsynonymous) and Ks (synonymous)
substitution rates of gene duplication pairs were calculated using
the “Simple Ka/Ks Calculator”function of TBtools. Ka/Ks <1, = 1,
and >1 represent purification selection, neutral selection, and
positive selection, respectively (Zhang et al., 2006). The
divergence time (million years ago/MYA) was calculated through
formula T = Ks/2l*10
−6
(l= 6.5 × 10
−9
).
2.4 Protein structure and subcellular
localization prediction
Secondary structure prediction of MsYUCs was performed by
Phyre2 (http://www.sbg.bio.ic.ac.uk/servers/phyre2/html/page.cgi?
id=index). A tertiary structure model of the MsYUC proteins was
predicted by SWISS-MODEL (https://swissmodel.expasy.org//).
Global model quality estimation (GMQE) was used to obtain the
high score-predicted model. Trans-membrane domain (TMD)
prediction was constructed using TMHMM based on the hidden
Markov model (https://services.healthtech.dtu.dk/services/
TMHMM-2.0/). Using the online website CELLOv.2.5, subcellular
localization was predicted (http://cello.life.nctu.edu.tw/ )(Yu
et al., 2010).
2.5 Analysis of the promoter-based cis-
acting elements
Promoter sequences of the MsYUC genes (2,000 bp upstream of
the ATG) were extracted by the “GTF/Gff3 Sequence Extract”
function of TBtools using “genome annotation file”and “genome
fasta file.”The promoter sequences were submitted to the
PlantCARE (http://bioinformatics.psb.ugent.be/webtools/
plantcare/html/) website for cis-acting element analysis, and the
elements represented by different-colored symbols were visualized
using TBtools’“Basic Biosequence viewer”function.
Shao et al. 10.3389/fpls.2023.1268027
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2.6 Analysis of MsYUC gene expression
patterns in the RNA-seq data
RNA-seq data of different tissue were downloaded from the
online LegumeIP V3 website (https://www.zhaolab.org/LegumeIP/
gdp). The expression data of different genotypes and under various
abiotic stresses were obtained from previous studies (Zhou et al.,
2018;Luo et al., 2019). The different expression profiles were
exhibited through a heat map constructed by “Amazing
Heatmap”function of TBtools.
2.7 RT-qPCR analysis
Eight-week-old seedlings of Zhongmu No. 1 were exposed to
untreated control (CK), cold (4°C), and salt (200 mM NaCl) stresses
for 6 h. After treatment, RNeasy Kit (Qiagen) was used to extract
the total RNA from three biological replicates under control, salt,
and cold stresses, respectively. First-strand cDNA of each sample
was synthesized using the TaqMan reverse transcription kit
(Applied Biosystems). qPCR was conducted on an ABI real-time
PCR system with a total volume of 20 ml containing 10 ml of SYBR
Green real-time PCR master mix (Toyobo, Japan), 2 ml of cDNA
template, 0.2 mM of upstream and downstream primers. The qPCR
program was conducted with denaturation at 95°C or 10 min,
followed by 40 cycles of amplification (95°C for 30 s, 60°C for 30 s,
and 68°C for 1 min) using the ABI real-time PCR system (Applied
Biosystems, Foster City, CA). Transcript levels of each sample were
determined and normalized to the untreated control sample (CK) as
a calibrator with respect to the internal control gene using the
2
−DDCt
method (Schmittgen and Livak, 2008). Values represent
mean ± SD of three biological replications. One-way ANOVA test
was used, and significant differences from CK and treated plants at
P< 0.05 are shown by asterisks. All the technical aspects of qPCR
experiments fitted the MIQE Guidelines (Bustin et al., 2009). The
primers used are listed in Table S1.
2.8 Protein–protein interaction and miRNA
target prediction
All MsYUC protein sequences were submitted to the STRING
website (http://string-db.org) to build a protein–protein interaction
network with their Arabidopsis orthologs as a reference. Using M.
truncatula miRNAs as reference, target miRNAs were predicted
through the psRNATarget website (https://www.zhaolab.org/
psRNATarget/) with default parameters while selecting target
accessibility, as previously described (Dai et al., 2018).
3 Results
3.1 Identification and basic characterization
of the MsYUC and MtYUC gene families
Comparative homology analysis was performed using the
downloaded Arabidopsis YUC protein sequences as Query to
search the protein sequences and the genome sequence of
Medicago, and a total of 12 MsYUCs and 15 MtYUCs were
identified from M. sativa and M. truncatula, respectively. All
members were designated MsYUC1-MsYUC12 and MtYUC1-
MtYUC15 according to their distribution and location
information on the chromosome (Table 1;Figure S1). The
MsYUC genes showed a significant uneven distribution on eight
chromosomes, with the most four MsYUC genes on chromosome 1,
three MsYUC genes on chromosomes 3 and 7, and only one MsYUC
gene on chromosomes 5 and 6, but no distribution of MsYUC genes
on chromosomes 2, 4, and 8 (Figure S1A). However, MtYUC genes
TABLE 1 Characteristics of the YUC gene family members in Medicago.
ID Name ORF Start End W/Da pI Location
MsG0180001906.01 MsYUC1 423 29138325 29139895 47167.58 9.12 Periplasmic
MsG0180002563.01 MsYUC2 385 40447849 40451427 43510.12 8.67 Cytoplasmic
MsG0180002571.01 MsYUC3 385 40561568 40564952 43526.16 8.78 Cytoplasmic
MsG0180003762.01 MsYUC4 420 67805768 67807662 47025.45 9.01 Cytoplasmic
MsG0380016438.01 MsYUC5 527 83416606 83420598 59400.64 8.98 Periplasmic
MsG0380016439.01 MsYUC6 360 83450071 83452625 40965.77 9.1 Periplasmic
MsG0380017591.01 MsYUC7 425 98226131 98230092 47542.93 8.82 Cytoplasmic
MsG0580025734.01 MsYUC8 511 23344641 23350209 57393.75 8.63 Periplasmic
MsG0680035661.01 MsYUC9 573 110273357 110282037 64111.41 8.78 Cytoplasmic
MsG0780040831.01 MsYUC10 416 83110763 83112388 46856.19 8.62 Cytoplasmic
MsG0780041255.01 MsYUC11 419 88839687 88843914 47701.96 8.7 Cytoplasmic
(Continued)
Shao et al. 10.3389/fpls.2023.1268027
Frontiers in Plant Science frontiersin.org04
were distributed in all chromosomes except for chromosome 2
(Table 1;Figure S1B). Chromosomal localization also showed that
all YUC genes could be localized to the Medicago chromosomal
genome. As shown in Table 1, the length of the coding region (ORF)
of MsYUC genes varied from 360 to 573 amino acids with a
molecular weight (MW) from 40.97 to 64.11 kD. The ORF and
MW of MtYUC genes varied in relatively small ranges, with The
ORF from 382 to 430 amino acids and MW from 42.87 to 48.28 kD
(Table 1). All YUC proteins were basic proteins with isoelectric
points (pI) greater than 8 (ranging from 8.1 to 9.12). Subcellular
location prediction showed that both MsYUC and MtYUC proteins
had cytoplasmic and periplasmic locations (Table 1).
3.2 Phylogenetic analysis of
Medicago YUC proteins
To further analyze the kinship of YUC genes, YUC protein
sequences from M. sativa (12 MsYUC), Arabidopsis (11 AtYUC),
M. truncatula (15 MtYUC), and rice (14 OsYUC) were selected, and
an evolutionary tree was constructed (Figure 1A). The results
showed that 52 YUC proteins in four species can be clustered
into two large clusters (clade I and clade II). Clade I can be further
subdivided into five small clades, with MsYUC1, 4, 10 and
MtYUC3, 4, 12 in clade I-1, MsYUC7, 9 and MtYUC1, 8, 11 in
clade I-2. Clade II can be subdivided into four small clades, with
MsYUC2, 3, 11, 12 in Clade II-2, MsYUC8, 5 and 6 in clade II-4.
MtYUC2 showed a close relationship with AtYUC1 and AtYUC4,
which were clustered into clade I-4. MsYUC7/9 and MtYUC1/8/11,
belonging to clade I-2, were relatively closely related to AtYUC6.
MtYUC10, MtYUC15, and MsYUC8 were closely related to
AtYUC10, which belongs to clade II-4 (Figure 1A). A
phylogenetic tree of four species was constructed, and the number
and distribution of YUC proteins in various subfamilies in four
species were counted (Figure 1B). Notably, clade I-3 and cladeII-3
only contained YUC proteins from rice and clade I-4 only
contained YUC proteins from the other three species except M.
sativa. Clade II-2 only contained YUC members from Medicago
with no homologous gene from Arabidopsis. Clade II-4 contained
YUC proteins from the other three species except rice. Only two
clades, clade I-1 and clade I-2, both contained YUC proteins from
the four species (Figure 1C).
3.3 Gene duplication, synteny, and
evolution analysis of the YUCs
Tandem and segmental duplication events were analyzed to
further investigate the evolutionary pattern of the YUC gene family
in Medicago. Results revealed that MsYUC5/MsYUC6 on
chromosome 3 and MsYUC11/MsYUC12 on chromosome 7 were
obvious tandem duplication genes (Figure S1A). Only one MsYUC
gene pair (MsYUC4/MsYUC10) could be identified as segmental
duplication events (Figure 2A). The MtYUC gene family has an
additional tandem repeat gene pair on chromosome 3 (MtYUC5/6,
MtYUC6/7)(Figure S1B;Table S1). Only one MtYUC gene pair
(MtYUC1/MtYUC8)ofM. truncatula was identified as segmental
duplication genes (Figure 2B). Comparative syntenic maps of M.
sativa with Arabidopsis, rice, and M. truncatula were constructed to
illustrate the evolution relationship of the YUC gene family
TABLE 1 Continued
ID Name ORF Start End W/Da pI Location
MsG0780041256.01 MsYUC12 383 88846915 88850153 43072.34 8.1 Periplasmic
AES58795 MtYUC1 430 883915 889499 48280.0 8.87 Cytoplasmic
AES58948 MtYUC2 406 2133963 2136290 45687.8 8.91 Periplasmic
KEH41176 MtYUC3 423 17407546 17409111 47142.53 9.12 Periplasmic
KEH42432 MtYUC4 421 29855829 29858303 47130.5 8.95 Cytoplasmic
KEH35392 MtYUC5 398 40705084 40707031 44902.85 8.38 Periplasmic
KEH35393 MtYUC6 391 40711936 40713932 44194.08 8.73 Periplasmic
KEH35394 MtYUC7 398 40720455 40722348 44929.91 8.38 Cytoplasmic
AES73853 MtYUC8 423 50666906 50671263 47154.53 8.81 Cytoplasmic
KEH29783 MtYUC9 399 18624417 18627401 45407.79 8.99 Cytoplasmic
AES96101 MtYUC10 382 14352361 14354443 42872.11 8.7 Cytoplasmic
KEH27129 MtYUC11 408 32789500 32793211 45302.98 9.1 Cytoplasmic
AES81674 MtYUC12 416 39838750 39840931 46783.18 8.72 Cytoplasmic
KEH24362 MtYUC13 383 43925708 43928430 43201.76 8.95 Cytoplasmic
KEH24364 MtYUC14 384 43937223 43940456 43148.61 8.7 Periplasmic
KEH18954 MtYUC15 382 12379792 12381700 42913.26 8.42 Cytoplasmic
Shao et al. 10.3389/fpls.2023.1268027
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(Figure 2C). Notably, 10, 10, and 1 orthologous pairs were found
between M. sativa and Arabidopsis,M. sativa and M. truncatula,
and M. sativa and O. sativa, respectively (Figure 2C). The collinear
blocks in which MsYUC4, 5, 7, 9, 10 were located is present in M.
truncatula and Arabidopsis except rice. MsYUC1-related collinear
blocks were found only in Medicago but not in Arabidopsis or rice
(Table S2). Interestingly, one MsYUC family member, MsYUC9,
had collinear relationships with gene(s) in all species analyzed
(Table S2). The Ka/Ks ratio of homologous MsYUC gene pairs
ranged from 0.19 (MsYUC4/10) to 0.49 (MsYUC5/6), whereas the
Ka/Ks ratio of MtYUC homologous ranged from 0.09 (MtYUC5/6)
to 0.24 (MtYUC6/7), indicating that the YUC genes of Medicago had
undergone a great purification selection pressure (Table S3). The
evolutionary divergence time (MYA) calculated showed that two
homologous gene pairs MsYUC4/10 (47.83 MYA) and MtYUC1/8
(57.28 MYA) were derived from the formation period of genus
Medicago.ThreegenepairsofMsYUC5/6,MtYUC5/6,and
MtYUC6/7 homologous gene pairs were derived around 5 million
years ago, and one homologous gene pair (MsYUC11/12)was
derived around 75.77 MYA (Table S3).
3.4 Motif and gene structure analysis of
MsYUC members
Motif analysis showed that 12 MsYUC proteins all contained the
conserved FAD-binding motif and NADPH-binding motif
(Figure 3A), suggesting a conserved function. Nevertheless, some
BC
A
FIGURE 1
The phylogenetic analysis and subfamily clusters of YUC proteins in plants. (A) Phylogenetic analysis using YUC proteins in Medicago (MsYUC and
MtYUC), Arabidopsis (AtYUC), and rice (OsYUC). The phylogenetic tree was constructed using the ClustalX program and the neighbor-joining
method. (B) Evolutionary relationships among four species. Phylogenetic analyses of four species were performed using “phyliptree.phy”from the
NCBI Taxonomy function. (C) Number of YUC proteins in four species and their distribution in various subfamilies.
Shao et al. 10.3389/fpls.2023.1268027
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differences were observed in the FMO-identifying motif of MsYUC5/6/
10 proteins, ATG-containing motif1 of MsYUC2/3/9/11/12 proteins,
and ATG-containing motif2 in MsYUC2/3/11/12 proteins, which
might contribute to the functional divergences (Figure 3A). In the
prediction analysis of the conserved motif of MsYUC proteins, 12
relatively conserved motifs (motifs 1~12) were further identified,
including motif1 as the FAD binding site and motif2 as the reduced
NADPH binding site (Table S4;Figure 3B). Furthermore, eight
conservedmotifs(motif1,2,4,5,6,8,9,10)werepresentinall
MsYUCs examined. Each MsYUC protein contained a minimum of 8
to a maximum of 12 of these motifs. and MsYUC6 protein had the least
motif. MsYUC1 and MsYUC4 protein had all 12 conserved motifs and
MsYUC7 had 11 conserved motifs except motif12. Seven MsYUC
proteins (MsYUC9/12/11/2/3/8/5) contained the same 10 conserved
motifs (motif1~10). Only MsYUC10 protein lacked motif8 compared
with other members (Figure 3B). Gene structure analysis revealed that
the number of exons of MsYUCs varied from 3 to 7 whereas MsYUC9
contained the most numerous introns. Five MsYUC genes (MsYUC2/3/
5/8/11)hadfive exons. Three MsYUC genes (MsYUC7/12/6)hadfour
exons, and three MsYUC genes (1/4/10) had three exons. Seven
MsYUC genes (MsYUC12/11/2/3/8/5/6) containing the same 10
conserved motifs had four exons, whereas three members had only
two exons (MsYUC1/4/10)(Figure 3C).
3.5 Expression analysis of MsYUCs in
different tissues and different genotypes
The expression patterns of 12 MsYUC genes in different tissues
were examined using online transcriptome data. Results indicated a
tissue expression specificity of different MsYUC genes. For example,
MsYUC10,MsYUC12,andMsYUC2 had relatively higher
expression levels in specific tissues examined, whereas some
members (MsYUC5/6/8) had very low expression levels and were
barely detectable. In addition, MsYUC2 had a higher expression
level in leaves than in other tissues and MsYUC12 was more highly
expressed in both leaves and roots than in other tissues (Figure 4A).
In addition, we further analyzed the expression correlation between
every two MsYUC genes in five tissues. MsYUC2 showed to be
significantly positively correlated with MsYUC3, consistent with
their close relationship in the phylogenic tree. MsYUC2 and
MsYUC3 showed to be significantly positively correlated with
MsYUC4 and MsYUC7, respectively. MsYUC3 showed to be
significantly positively correlated with MsYUC4 and MsYUC7,
respectively. MsYUC4 and MsYUC7,aswellasMsYUC1 and
MsYUC9,weresignificantly positively correlated (Figure 4B).
There was also a differential expression pattern of MsYUCs
among different genotypes. For example, MsYUC10 and
B
C
A
FIGURE 2
Duplication event analysis for the YUC gene family in the M. sativa (Ms) and M. truncatula (Mt) genome and synteny analysis between M. sativa and
the other three species. The duplication events in the M. sativa genome (A) and M. truncatula genome (B). Red-colored lines indicate duplication
events of MsYUC family members (MsYUC10/4) and MtYUC family members (MsYUC1/8). (C) Collinearity analysis of M. sativa (Ms) with M. truncatula
(Mt) or Arabidopsis (At) or rice (Os). Red-colored lines indicate the YUC family members in different species.
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MsYUC12 showed a higher expression level in 95-608 compared
with other genotypes. MsYUC11 had a relatively higher expression
level in PI251830-K but had the lowest expression detected in 95-
608 (Figure 4C).
3.6 Promoter analysis and stress response
expression of MsYUCs
To predict the possible regulation of MsYUCs expression, the
cis-acting elements included in the promoter sequence of the
MsYUC genes were analyzed. Results revealed a variety of stress
response elements related to hormone and stress response
(Figure 5A). Auxin-responsive elements were found in the
promoter region of MsYUC1,4, 5, 8, 12,andthreeoftheir
promoters contained AuxRR-core elements. The promoter of
MsYUC8 had the most cis-acting elements (6) involved in the
abscisic acid responsiveness (ABRE) (Figure 5B). The MsYUC7
promoter region contained five CGTCA motifs, which functions in
Me-JA responsive. There were also some GA-responsive elements
such as GARE-motif, TATC-box, P-Box, and some SA-responsive
elements (TCA-element) in the promoters of certain MsYUCs
(Figure 5A). The promoter of MsYUC6,8, and 10 contained one
cis-acting element involved in low-temperature responsiveness LTR
(CCGAAA), respectively. Except for MsYUC6,7,and8,other
members all had one or two MYB-binding site (MBS) involved in
drought inducibility. Some anaerobic induction, osmotic pressure-
responsive, and defense and stress-responsive elements (TC-rich
repeats) were also present on certain MsYUC promoters
(Figure 5A). In addition, all the MsYUC promoters had light-
response elements. MsYUC1, 2, 8 had the most light-response
elements (6) whereas MsYUC12 had the least light-response
element (1) (Figure 5B). Based on the abiotic transcriptome data
analysis, the expressions of MsYUC1 and MsYUC10 were
significantly increased under salt stress (Figure 5C)andthe
expressions of MsYUC10 and MsYUC12 were induced by cold
(Figure 5D). Mannitol treatment significantly induced the
expression of MsYUC10 (Figure 5E). RT-qPCR analysis further
confirmed that MsYUC1 and MsYUC10 expression could be
induced by NaCl (100 mM) and MsYUC10 and MsYUC12 could
be elevated by cold (4°C) for 3 h treatments (Figures 5F,G).
Expression analysis showed that MsYUC genes might have a
tissue-specific expression and differential abiotic stress
response pattern.
BC
A
FIGURE 3
Structure and conversed motifs of MsYUC members. (A) Alignment of conserved domains in MsYUC proteins. (B) Conserved motifs of MsYUC genes
predicted by MEME. (C) Gene structure of MsYUC genes. The exons are represented by blue boxes, and black lines are represented by black lines.
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3.7 Prediction of the protein interaction
network and targeted miRNA of
MsYUC members
Protein structure prediction showed that MsYUC proteins
shared a unique topology containing several a-helices and b-
structures (Figures 6A,S2), indicating structural conservation.
The trans-membrane prediction showed that MsYUC4, 9, 10, and
11 proteins possessed one TMD, respectively. The TMD regions of
MsYUC9 and 11 proteins were localized in the N-terminal, whereas
the TMD regions of MsYUC4 and 10 were localized in the middle of
the protein (Figure S3). A predicted protein interaction network
indicated that MsYUC proteins had multiple interaction partners
(Figure 6B). MsYUC10 protein was predicted to interact with
transcription factor NAC089 and NAC-like NTL9, and auxin
upregulated F-box protein 1 (AUF1), which is a component of E3
ubiquitin ligase complexes. Both MsYUC9 and MsYUC10 proteins
were predicted to interact with phytochrome interacting factor 4
(PIF4). MsYUC9 protein could also interact with TAA1 and
amidase 1 (AMI1), which functions in auxin biosynthesis.
MsYUC1, 7, 9, 10, 12 were predicted to interact with TAA1,
TAR1, and TAR2, which function in the first step of the IPA
pathway. We next performed miRNA target site prediction for the
MsYUC genes. As shown in Figure 6C,MsYUC2, 3, and 11 were
predicted to be targeted by a similar miRNA5272f. MsYUC5, 6, and
8were predicted to be targeted by a similar miRNA5742. All the
coding sequences of MsYUCs contained at least three predicted
targets for miRNA.
4 Discussion
The YUC gene family proteins involved in auxin biosynthesis
are the first identified FMO class family in plants that regulate
growth, development, and tolerance in plants (Zhao et al., 2001). In
Medicago, the YUC number (12 MsYUCs and 15 MtYUCs) was
close to 11 AtYUC in Arabidopsis and 14 OsYUC in rice (Yamamoto
et al., 2007;Zhao, 2012). Gene duplication is thought to be the main
driver of species evolution and a direct cause of gene family
expansion (Lynch and Conery, 2000;Moore and Purugganan,
2003;Maere et al., 2005), and two forms of gene duplication
(tandem and segmental) events were identified in Medicago YUC
gene families. In the MtYUC gene family, there was a gene cluster
containing three MtYUC members (MtYUC5/6/7). Moreover, there
was no distribution of MsYUC genes on chromosomes 2, 4, and 8
(Table 1,Figure S1A) whereas MtYUC genes were distributed in all
chromosomes except for chromosome 2 (Table 1;Figure S1B).
These reasons may together contribute to the more members of
MtYUC than that of MsYUC. Notably, most of the YUC proteins in
rice (Monocots) and Medicago or Arabidopsis (Dicots) could not
gather under the same branch, as clade II-4 had no rice YUC
protein and clade I-3 and clade II-3 only contained rice YUC
B
C
A
FIGURE 4
Expression pattern of MsYUC genes in different tissues and in different genotype. (A) Tissue-specific expression analysis of MsYUC genes. (B) The
correlation of gene expression patterns between every two MsYUC genes. Red and blue circles represent positive and negative correlations,
respectively. (C) MsYUC expression in different genotypes. The color scale of the heatmap refers to the relative expression level.
Shao et al. 10.3389/fpls.2023.1268027
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proteins, indicating that YUC proteins underwent an evolutionary
divergence, as that there are missing or duplication of YUCs during
evolution. (Figure 1). In addition, Ka/Ks was used to evaluate their
specific positions under positive selection pressure after duplication
(Lynch and Conery, 2000;Mayrose et al., 2007). In this study, the
Ka/Ks value of each duplication gene pair of YUCs of Medicago for
all gene pairs was less than 1 (Table S3), which suggested that these
genes had evolved under strong purifying selection. Since the
divergence time of the Papilionoideae subfamily, which includes
the genus Medicago, was approximately 34–63.7 millions of years
(MYA) (Wang et al., 2023), the evolutionary divergence time of
homologous gene pairs MsYUC4/10 and MtYUC1/8 was derived
from the formation period of Papilionoideae subfamily. Because of
the importance of M. sativa with high yield, nutrient value, and
palatability, the mechanisms regulating its growth are of significant
interest (Yamamoto et al., 2007). Functional orthologs of YUC
genes in model species can provide insight into the functions in
Medicago (Wei and Gai, 2008). MtYUC2 showed a close
relationship with AtYUC1 and AtYUC4 (Figure 2), which have
been reported to play vital roles in the formation of floral organs
and vascular tissues in Arabidopsis (Cheng et al., 2006). However,
the Arabidopsis AtYUC1 and AtYUC4 had no corresponding
homologs in the M. sativa genome. Moreover, some YUCs of M.
sativa had no homologs in Arabidopsis or rice, indicating that the
gene loss event may have occurred after species divergence
(Figure 1;Table S2).
In Arabidopsis, the roots and shoots appear to use two separate
sets of YUC genes for auxin biosynthesis: ER-located YUCs
functioning in roots or cytoplasmic-located YUCs functioning in
shoots (Kriechbaumer et al., 2015). Phylogenetic tree analysis
showed that AtYUC3, 5, 7, 8,and9, which were reported to
function in roots with ER location, clustered in clade I-1
(Kriechbaumer et al., 2015). MsYUC4 and MsYUC10, closely
related to AtYUC5, 8, 9, also showed a predicted cytoplasmic
location (Table 1). In M. sativa,MsYUCs also showed different
expression patterns in different tissues. For example, MsYUC10,
B
CD
E
F
A
G
FIGURE 5
Cis elements of MsYUC promoter prediction and expression analysis of MsYUCs under stress conditions. (A) Number of hormone and stress
response-related elements of MsYUCs.(B) Main elements distributed in the promoter region of MsYUC genes. Expression of MsYUC genes under
salt stress (C), mannitol treatment (D), and cold stress (E). The color scale of the heatmap refers to the relative expression level. Relative expression
of MsYUCs treated by NaCl (F) and cold (G) determined by RT-qPCR. Three replicates were designed for each sample, and M. sativa actin gene
expression was used for data normalization. Value represents mean ± SD of three replicates. * indicated significant different from untreated control
(CK) plants (p < 0.05, one-way ANOVA).
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MsYUC12, and MsYUC2 had relatively higher expression levels in
specific tissues examined and MsYUC2 had a higher expression
level in leaves than in other tissues (Figure 4A). MsYUC9, closely
related to AtYUC2, which was reported to function in shoots, was
expressed not only in the shoots but also in roots of M. sativa.
MsYUC12, with no homologous genes in Arabidopsis,showed
higher expression levels in all tissues and was inferred to have
universal roles during plant growth and development (Figure 5A).
Therefore, in contrast to AtYUC expression, MsYUC expression
does not seem to be clearly divided into shoot or root independent
expression, suggesting a specificity in M. sativa compared
with Arabidopsis.
The IPA-dependent pathway also plays an important role in
integrating environmental stress and hormone signaling, and YUCs
were reported to be involved in environmental stress response
(Blakeslee et al., 2019). Cis-acting elements on the MsYUCs’
promoter revealed a variety of stress response elements related to
hormone such as Auxin-, ABA-, JA-, GA-, and SA-responsive
elements in the promoters of certain MsYUC genes (Figure 5A).
In Arabidopsis, ABA can inhibit the transcription of YUC2/8 via
ABI4, thereby inhibiting primary root elongation (Yu et al., 2014).
JA has been reported to promote lateral root growth through a
direct regulation of YUC2 by transcription factor ERF109 (Cai et al.,
2014). JA also directly activates YUC8/9-dependent auxin
biosynthesis to function in mechanical wounding response
(Perez-Alonso et al., 2021). In M. sativa, six ABRE elements were
found in the promoter of MsYUC8 and five JA response elements
were found in the promoter of MsYUC7, respectively. In addition,
MsYUC7 showed a closer relationship with AtYUC2, implying a
similar function in JA response. In Arabidopsis, expression levels of
YUC7, 9, 10, and 11 were upregulated under dehydration conditions
(Shi et al., 2014). Activation of YUC7 enhances drought resistance
in Arabidopsis (Lee et al., 2012). Overexpressed YUC6 of
Arabidopsis in potato and poplar plants or overexpressed
BnaYUC6a in Arabidopsis and oilseed rape showed typical auxin
overproduction alternation and conferred high drought resistance
(Kim et al., 2012;Ke et al., 2015;Hao et al., 2022). Since MsYUC11,
which is closely related to AtYUC6, had a five-element response to
osmotic stress but no drought response elements, suggesting a
function differentiation among species (Figure 5A).
YUC expression was also reported to be affected by cold stress. For
example, cucumber CsYUC10b was upregulated by cold stress whereas
other CsYUCs were downregulated (Yan et al., 2016). In the hypocotyl,
the PIF4-YUC8 regulatory module plays an important role in response
to stress signals, including light stress. The accumulation and
transcriptional activity of PIF4 are regulated by different proteins,
with competition for and interference at the AtYUC8 promoter by
other transcription factors affecting the positive regulation of AtYUC8
BC
A
FIGURE 6
Predicted protein interaction network of MsYUC proteins and miRNA target sites in MsYUC genes. (A) Protein structure prediction of MsYUCs.
(B) Protein interaction network predicted using MsYUC orthologs from Arabidopsis.(C) Predicted miRNA targets in the MsYUC coding sequence. The
red tangles represent the miRNA-targeted MsYUC sites.
Shao et al. 10.3389/fpls.2023.1268027
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by PIF4 and consequently affecting biosynthesis of auxin (Ma et al.,
2016). In this study, all the MsYUC promoters had light-response
elements. MsYUC1, 2,and8promoters had the most light-response
elements, whereas the MsYUC12 promoter had the least light-response
element (Figure 5A). MsYUC10 was further predicted to interact with
PIF4, indicating a similar function with AtYUC8 in light response
(Figure 6B). Transcription factor AGL21 positively regulates AtYUC5/8
which could be induced by IAA/ABA/JA and a variety of stresses,
including salt stress (Yu et al., 2014). MsYUC1 and MsYUC10, which
were clustered in the same sub-clade with AtYUC3/5/7/8/9, showed a
significantly salt-induced expression (I-1), indicating a salt-response
function in M. sativa (Figure 5F). The promoter of MsYUC1, 2,and3
contained one cis-acting element (LTR) involved in low-temperature
responsiveness, respectively (Figure 5A). Cold stress significantly
elevated the expression of MsYUC10 and MsYUC12, indicating an
LTR-independent cold stress response function (Figure 6B). Moreover,
MsYUC10 and MsYUC12 showed a higher expression level in 95-608
compared with other genotypes. Therefore, the stress tolerance of 95-
608 should be further compared with other varieties. Studies indicate
that miRNA-directed regulation of transcription factors may also play
key roles in the precise regulation of IPA-dependent auxin biosynthesis
in plants (Luo and Di, 2023). In this study, all MsYUCs contained at
least three predicted targets for miRNA, suggesting a miRNA-directed
regulation of YUC in M. sativa (Figure 6C).
5 Conclusion
In this study, the YUCs of M. sativa and M. truncatula were
identified on a genome-wide scale. The phylogenetic analysis and
comparative syntenic maps of M. sativa with other species
illustrated their evolution relationship. The tissue and genotype-
specific expression and abiotic stress response profiles have also
been analyzed to reveal potential functional YUC genes. Moreover,
RT-qPCR verified that certain MsYUC members represented salt or
cold stress-affected expression patterns. Results in this study could
provide valuable information for functional analysis and for the
underlying regulation mechanism study of a specificMsYUC gene
of M. sativa, especially under different tissues and various abiotic
stresses through modification of the auxin synthetic IPA pathway
in Medicago.
Data availability statement
The original contributions presented in the study are included
in the article/Supplementary Material. Further inquiries can be
directed to the corresponding authors.
Author contributions
AS: Conceptualization, Data curation, Methodology, Writing –
original draft. SF: Methodology, Software, Writing –original draft.
XX: Validation, Investigation, Writing –review & editing. WW:
Funding acquisition, Resources, Writing –review & editing. JF:
Project administration, Supervision, Writing –review & editing.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This work
was supported by grants from the National Natural Science
Foundation of China (No. 32001389) and the Natural Science
Foundation of Shandong Province, China (No. ZR2020QC186).
Acknowledgments
We thank the National Natural Science Foundation of China
and the Natural Science Foundation of Shandong Province for the
financial support.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1268027/
full#supplementary-material
Shao et al. 10.3389/fpls.2023.1268027
Frontiers in Plant Science frontiersin.org12
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