Molecular Biology Reports (2021) 48:1269–1279

https://doi.org/10.1007/s11033-021-06197-0

ORIGINAL ARTICLE

Genome‑wide identification and expression analysis of the TaYUCCA​

gene family in wheat
Yanlin Yang1 · Tian Xu1 · Honggang Wang1 · Deshun Feng1 

Received: 23 October 2020 / Accepted: 28 January 2021 / Published online: 5 February 2021
© The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021

Abstract
Auxin is an important endogenous hormone in plants. The YUCCA​ gene encodes a flavin monooxygenase, which is an
important rate-limiting enzyme in the auxin synthesis pathway and involved in the regulation of plant growth and develop-
ment. In the study, we identified 63 wheat TaYUCCA​genes; among them, some genes appeared in clusters. By constructing
phylogenetic trees, we found that the TaYUCCA​genes could be divided into six groups. In the WheatExp database, there were
22 differential expressed TaYUCCA​genes, among which the TaYUCCA10 gene was abundantly expressed in the endosperm
and medium milk stage, the TaYUCCA2 gene was abundantly expressed in the roots of three leaves and meiosis and transfer
cells at 20 days post anthesis and the others 16 TaYUCCA​ genes had different expression level at different developmental
stages in wheat, and there were 15 TaYUCCA​ genes induced by drought and heat stress, among which the TaYUCCA2-D,
TaYUCCA3-B, and TaYUCCA9-D might be upregulated induced by drought stress, TaYUCCA10.1 might be upregulated
induced drought and heat stress, TaYUCCA6-A was upregulated induced both drought and heat stress and the others 9 TaY-
UCCA​ genes were downregulated induced by drought and heat stress. Transcriptome and qRT-PCR analysis showed that
TaYUCCA7-A was upregulated significantly after induced by powdery mildew. The comprehensive annotation and expression
profiling of the TaYUCCA​ genes in this study enhanced our understanding of TaYUCCA​family gene expression in wheat
growth and development and laid the foundation for the further study of TaYUCCA​gene mechanism.

Keywords Wheat · TaYUCCA​ · Phylogenetic analysis · Expression profiles

Introduction gene family is involved in the Trp-dependent pathway in the

Auxin is an endogenous hormone that plays a vital role in
plant growth and development, such as fruit development
[1], lateral root formation [2], leaf morphology [3], and
flower development [4]. In addition, auxin participates in
the response to abiotic and biotic stresses, such as salt [5],
drought [6], cold stresses [7], and pathogen invasion [8].
Indole-3-acetic acid (IAA) is mainly synthesized in the
shoot apices and tips and can be transported across the mem-
brane from one cell to another [9]. Two major pathways for
IAA biosynthesis, namely, the Trp-dependent and Trp-inde-
pendent pathways [10], have been proposed. The YUCCA​
* Deshun Feng
dsfeng@sdau.edu.cn
1
State Key Laboratory of Crop Biology, Shandong Key
Laboratory of Crop Biology, College of Agronomy,
Shandong Agricultural University, Tai’an 271018,
Shandong, China
auxin synthesis pathway and includes four branches. Trp is
first converted to indole-3-pyruvate (IPA) by the TAA family
of amino transferases. Subsequently, the flavin monooxy-
genase (FMO) encoded by the YUCCA​ gene catalyzes the
conversion of IPA to IAA [11]. The two-step conversion of
Trp to IAA is the main auxin biosynthesis pathway that plays
an essential role in many developmental processes [12].
FMO was initially found in liver microsomes; it can
transfer hydroxyl groups to atoms of insoluble nucleophilic
compounds, resulting in increased polarity and solubility
and accelerated the excretion of insoluble compounds [13].
Hou et al. demonstrated that the conserved domain of FMO
contains two conserved motifs (FAD and NADPH bind-
ing motifs), which have the same G × GxxG characteristic
structure in their amino acid sequences [14]. The FMO-
identifying motif (FxGxxxHxxxY/F) is the key sequence
in all known plant FMOs and can help us identify FMO in
the protein databank [15]. It was reported that the genome
of the model plant Arabidopsis thaliana contains 29 genes

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coding for proteins with significant sequence similarity to
human FMOs [16]. These 29 FMO genes have been divided
into three clades [17]. Clade II contains the 11 Arabi-
dopsis YUCCA​ genes involved in the biosynthesis of the
plant hormone indole-3-acetic acid [11] and the 11 YUC​
genes have overlapping functions during development [18].
Among these genes, AtYUC1, AtYUC2, AtYUC4, AtYUC6,
AtYUC10, and AtYUC11 play essential roles in auxin bio-
synthesis and plant development [18, 19]. Transgenic poplar
expressing AtYUCCA6 exhibited rapidly form bud growth,
main root development retardation, and increasing root hair
formation under abiotic stress [20]. Yamamoto et al. have
identified seven YUCCA​ genes in rice and found that OsY-
UCCA1 was expressed in almost all organs tested, and its
overexpression increased the IAA levels and a phenotype of
auxin overproduction [21]. Exactly 22 YUCCA​ genes were
found in soybeans, and transgenic GmYUCCA5 A. thaliana
plants showed downward curling of the leaf edges, distinct
apical dominance, high plant height, and short length of
siliques [6]. Li et al. identified 14 YUCCA​ genes in maize
and found that the ZmYUCs might be involved in both veg-
etative and reproductive development [22].
With the completion of wheat genome sequencing and
the improvement of wheat genome databank, we systemati-
cally performed comprehensive identification in this study,
analyzed the detailed characteristics of 63 wheat TaYUCCA​
genes, and investigated their structures, functions, and the
expression level. This study lays a foundation for future
research about the cloning and biological functions of wheat
YUCCA​gene. This research is conducive to the application
of these genes in wheat genetic improvement.
Materials and methods
Identification of TaYUCCA genes in the wheat
genome
To identify the YUCCA​ gene in wheat, we performed
homologous alignment searches by using the YUCCA​
sequences of A. thaliana (AtYUCCA1-11), Triticum urartu
(TuYUCCA1-11), and rice (OsYUCCA1-8). The high confi-
dence protein sequences of wheat (iwgsc_refseqv1.0_High-
Conf_PROTEIN_2017Mar13) were downloaded to establish
a local Blast using BioEdit software. The obtained YUCCA
sequences from A. thaliana, Triticum urartu, and rice were
used to Blastp in the wheat database URGI (https​://wheat​
-urgi.versa​illes​.inra.fr/). The E value was set to the highest
(1.0E–100), and we analyzed the obtained high scores from
aligned sequence selection by comparison. Firstly, we get
621 candidate protein sequences. After deleted the repeated
sequences, we get 424 candidate protein sequences. The can-
didate sequences obtained from the preliminary screening
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Molecular Biology Reports (2021) 48:1269–1279
were subjected to gene structure analysis using the gene
structure prediction site Augustus (http://bioin​f.uni-greif​
swald​.de/augus​tus/). The obtained amino acid sequence was
then subjected to Pfam domain prediction in the SMART
website (http://smart​.embl-heide​lberg​.de/smart​/set_mode.
cgi?NORMA​L=1). The conservative Pfam domains of the
obtained amino acid sequences were volume predicated in
the websites of EMBL-EBI and HMMER, and the amino
acid sequences containing the FMO-like domain, which
contains two conserved motifs of FAD and NADPH were
reserved for further analysis.
Phylogenetic analysis of wheat TaYUCCA genes
We performed multiple sequence alignments of the identi-
fied YUCCA​sequences by using ClustalW and DNAMAN.
We performed clustering analysis by using the maximum
likelihood (ML) method based on the results of MEGA
10.1 matching comparison and repeated bootstrap tests for
1000 times. After converting the obtained cluster analysis
graph into nwk format, it was uploaded on the iTOL website
(http://itol.embl.de/) for drawing.
Analysis of gene and protein structure and their
organization
Data on the position of each YUCCA​ gene on the chromo-
some, start and termination position, sequence length, and
length of the open reading frame (ORF), were retrieved
from the annotation file iwgsc_refseqv1.0_HighConf_2017
Mar13 in the URGI database. The signal peptides were pre-
dicted using the SignalP website (http://www.cbs.dtu.dk/
servi​ces/Signa​lP/), and the gene structure map was gener-
ated using TBtools. Subcellular localization was predicted
using BaCelLo (http://gpcr.bioco​mp.unibo​.it/bacel​lo/index​
.htm), and the physicochemical properties of proteins, such
as isoelectric point (pI) and molecular weight (MW), were
predicted using the Compute pI/MW prediction online tool
in ExPASy (https​://www.expas​y.org).
The expression pattern in different stress
and different tissues
We used the published WheatExp database (https​://wheat​
.pw.usda.gov/Wheat​Exp/) to study the expression of TaY-
UCCA​genes in stress and in different tissues. The cDNA of
each member of the TaYUCCA​gene family was used as the
query sequence to search, the E value was set to (1.0E-10)
The relative expression of each TaYUCCA​ gene was pre-
sented as a heat map generated from the relative abundance
of transcripts (per 10 million reads) for each gene from the
WheatExp database [23].
Molecular Biology Reports (2021) 48:1269–1279 1271
Plant materials and treatment
Wheat cultivar Chinese Spring (Triticum aestivum L.) was
sown in mixed soil (peat moss, matrix, and vermiculite;
1:1:1, v/v/v) inside a plant growth climate chamber at 25 °C
with 70% humidity and a long sunlight photoperiod (8 h dark
and 16 h light). TaYUCCA​ transcriptome analysis induced
by powdery mildew was conducted as follows. When the 40
seedlings of wheat Chinese Spring grew to the three-leaf
stage, the spores of the powdery mildew physiological race
E09 was used for leaf spraying. After treatment for 0, 1, 2, 3,
4, and 5 days and three biological replicates were conducted,
the leaves were obtained and immediately placed in liquid
nitrogen at −80 °C before use.
For quantitative RT-PCR (qRT-PCR) analysis of TaYUC​
genes induced by drought and heat stress, total 40 Chinese
Spring wheat seeds were grown to the three-leaf stage by
hydroponics. They were treated with 20% PEG, high temper-
ature treatment (40 °C) and 20% PEG and high temperature
treatment (40 °C) co-treatment. The leaves were treated for
1 h and 6 h. They were collected and quickly placed in liquid
nitrogen. Each experiment was subjected to three biological
replicates and placed at −80 °C before use.
RNA extraction, cDNA synthesis, RNA‑seq,
and qRT‑PCR analysis
To study the TaYUCCAs’ expression of wheat induced
by powdery mildew, we used Chinese Spring inoculation
0–5 days of powdery mildew to construct a cDNA library
and the sequence via a paired-end strategy using the Illumina
HiSeq 2000 sequencing platform. Finally, the sequencing
data containing only clean reads were directly used for tran-
scriptome analysis [24]. The upregulated expression TaYUC​
CCA​genes were presented as a heat map generated from the
relative abundance of transcripts (per 10 million reads) for
each gene from the transcriptome database.
Total wheat RNA was extracted from the above wheat
leaves treated with powdery mildew, drought, heat, and
drought with heat by using the EasyPure® plant RNA kit
according to the manufacturer’s instructions. The total
RNA quality was evaluated using the Nano Photometer
P360 (IMPLEN, Germany). Total RNA samples were
used directly to synthesize the first cDNA by using Trans-
Script® First-Strand cDNA Synthesis SuperMix (Trans Gen,
Beijing, AT301-02). All qRT-PCR operations were carried
out using the ABI Quantitative PCR Q6 detection system.
Reactions were carried out under the following program:
94 °C for 30 s, 44 cycles: 94 °C for 5 s, and 60 °C for 30 s.
For the melt curve analysis, the following program was
added after 40 cycles: 95 °C for 10 s, 60 °C for 30 s, and
then a constant increase from 60 °C to 95 °C. Wheat Actin
gene was used as reference gene, in which three independent
biological replicates were conducted per analysis with at
least three technical replicates. Relative RNA expression
levels were determined using the 2­ −ΔΔCT method [25]. The
primers used to analyze the gene expression for qRT-PCR
were listed in Table S1.
Results
Identification and classification of wheat TaYUCCA
genes
Based on the domain predicted by Pfam, the 63 TaYUCCA​
genes encode proteins with FMO-like domain and contain-
ing the conserved binding motifs for FAD and NADPH were
finally identified. Among the 63 TaYUCCA​ genes, 24 TaY-
UCCA​genes are distributed on the A genome, 19 TaYUCCA​
genes are distributed on the B genome, and 20 TaYUCCA​
genes are distributed on the D genome (Fig. 1). In terms of
the distribution of homologous groups, there is no distribu-
tion loci was observed in the homologous group no. 6. The
maximum number of gene loci distributed in homologous
group no. 2 is up to 22, among them, chromosome 2B has
11 TaYUCCA​genes, representing the most abundant region.
Followed by chromosome 4A, which has 7 TaYUCCA​genes.
We found that some TaYUCCA​ genes appeared in clusters
on the chromosomes. Gene clusters result from gene tandem
duplication, and some TaYUCCA​genes are adjacent to each
other, thus forming gene clusters. The largest tandem repeat
gene cluster was found on chromosome 2B, which has eight
TaYUCCA​ genes members (Fig. 1). Tandem duplication is
common in plant chromosomes, and this process may help
plants evolve in the YUCCAs family and act under abiotic
and biotic stress. Information about the TaYUCCA​gene fam-
ily in the URGI database, including chromosomal location,
pI, MW, ORF length, gene length, and subcellular localiza-
tion of each gene are listed in Table S2.
Phylogenetic and structure analysis of YUCCA
from wheat, A. thaliana, and rice
The 63 TaYUCCA amino acid sequences were clustered via
the ML method by using MEGA 10.1. The results showed
that these TaYUCCA​genes could be divided into six groups.
Group I consisted of two genes, including TaYUCCA14-
A and TaYUCCA14-D, group II consisted of three genes,
including TaYUCCA9-A, TaYUCCA9-B and TaYUCCA9-D,
group III consisted of three genes, including TaYUCC4-
A, TaYUCC4-B, and TaYUCC4-D, group IV included six
genes, TaYUCC2-A, TaYUCC2-B, TaYUCC2-D, TaYUCC3-
A, TaYUCC3-B, and TaYUCC3-D, group V included nine
genes, TaYUCC1-A, TaYUCC1-B, TaYUCC1-D, TaYUCC8-
A, TaYUCC8-B, TaYUCC8-B2, TaYUCC8-D, TaYUCC17-A,
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Fig. 1  Distribution of 63 TaYUCCA​ genes on the wheat chromo-
somes. The chromosome numbers are indicated on the left side of
each chromosome. The genes placed on the right side of each chro-
and TaYUCC18-B. The remaining 40 genes were in group
VI (Fig. 2a).
To better understand the basis of sequence structure
selection in different stresses and functional differentia-
tion, we further analyzed the TaYUCCA protein conserved
motifs and found that the amino acid sequences in these six
groups contained conserved motifs, such as FAD-binding
motif (GxGxxG), FMO-like motif (FxGxxxHxxxY/F), and
NADPH-binding motif (GxGxxG) [17]. The group VI pro-
teins lacked ATG-containing motif 1 ­(Y(X)7ATGEN(X)5P)
and ATG-containing motif 2 (ATGY) compared with the
group I, II, III, IV and V proteins (Figs. S1, S2, S3, S4, S5,
and S6). In these five conserved motifs, the ATG-containing
motif 2 was located at the C-terminus of the entire amino
acid sequence, and the FAD-binding motif was located at the
N-terminus of the amino acid sequences. ATG-containing
motif 1, FMO-like motif, and NADPH-binding motif were
located in the middle region of the amino acid sequences,
and the three motifs were located close to each other. ATG-
containing motif 2 may link the NADPH site to the active
site [26].
The YUCCA amino acid sequences of dicotyledonous
A. thaliana, monocotyledonous rice, and common wheat
were clustered using MEGA10.1. Results showed that 63
YUCCA proteins could be divided into six groups based on
the classification of wheat YUCCA family (Fig. 2b). Among
these groups, group I contained OsYUCCA5 and two wheat
TaYUCCA proteins, group II contained OsCOW1 and three
wheat protein, group III contained two OsYUCCA proteins
OsYUCCA1 and OsYUCCA2, two A. thaliana YUCCA
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Molecular Biology Reports (2021) 48:1269–1279
mosome. The position (bp) and orientation of each TaYUCCA​ gene
on URGI chromosome are given in Table S2
proteins AtYUCC1 and AtYUCCA4 and three wheat pro-
tein, group IV contained two OsYUCCA, two A. thaliana
YUCCA, and six TaYUCCA proteins, group V contained
two OsYUCCA, five A. thaliana YUCCA, and nine TaY-
UCCA proteins, and group VI consisted of two AtYUCCA,
and 40 TaYUCCA proteins. The insertion of A. thaliana
and rice YUCCA​ genes broke the original large grouping
of wheat TaYUCCA​ genes into small, specific branches,
but the genes maintained the consistency of separate wheat
TaYUCCA​gene groupings. Moreover, the YUCCA​genes of
wheat had evolutionary relationship with A. thaliana and
rice YUCCA, indicating that the YUCCA​ gene is highly
conservative in evolution and providing important support
for further to analysis of the wheat YUCCA​genes (Fig. 2b).
Gene structure and evolutionary relationship
of wheat YUCCA​
To understand the function of the gene, we analyzed the
exons/introns of 63 TaYUCCA​ genes by using the exon/
intron analysis tool ESPRIPT (http://espri​pt.ibcp.fr/ESPri​
pt/cgi-bin/ESPri​pt.cgi). We found 0–8 introns in TaYUCCA​
genes, among which the TaYUCC12-D2 gene contained
eight introns, which is a TaYUCCA​ gene with the most
introns, whereas the other genes contained 0–4 unequal
introns. The total length of the YUCCA12-D2 gene was
approximately 13,338 bp. The longest intron of this gene was
approximately 5476 bp, and its ORF was 2196 bp. Most of
the genes contained four exons and three introns. Structural
Molecular Biology Reports (2021) 48:1269–1279 1273
Fig. 2  Phylogenetic tree analysis of YUCCAs. a Phylogenetic tree
and exon/intron structure of wheat TaYUCCA​ genes. The conserved
FMO-like domain which contains conserved motifs for binding of
FAD and NADPH of all wheat TaYUCCA proteins were aligned with
Clustal W, and the phylogenetic tree was constructed in MEGA 10.1.
The structure of all the TaYUCCA​ genes. Green boxes, straight lines
and yellow boxes represent exons, introns and untranslation region
(UTR), respectively. b Phylogenetic analysis of wheat, A. thaliana
and rice YUCCAs. The conserved FMO-like domains of all wheat
TaYUCCA proteins, 11 AtYUCCA proteins and 8 OsYUCCA pro-
teins were aligned using Clustal W, and the phylogenetic tree was
constructed in MEGA 10.1
diversity in gene families is one of the main drivers of multi-
gene family evolution.
According to the clustering results of the TaYUCCA​
genes, the structure of 63 TaYUCCA​genes was observed in
different groups. The results showed that the genes in group
I had the same gene structure and contained four exons and
three introns. In group II, the TaYUCCA9-B gene contained
four introns and five exons and exhibited different numbers
of introns than the TaYUCCA9-A and TaYUCCA9-D genes
(Fig. 2a). Multi-sequence alignment revealed that the cDNA
similarity among the three genes was 93.70%, and the simi-
larity of the protein sequence was 92.75%. In comparison
with the cDNA of TaYUCCA9-A and TaYUCCA9-D, the
cDNA of TaYUCCA9-B has a sequence of approximately
50 bp deletion at a position of approximately 200 bp, thus
possibly resulting in a gene structural diversity (Table S2).
In group III, the gene structures of the three genes are the
same. These genes all contain four exons and three introns,
and the number of amino acids is similar. TaYUCCA4-A
and TaYUCCA4-D have the same number of amino acids
(410 AA, Fig. 2a). The six genes in group IV have the same
gene structure as those in group III. The gene structure of
group V was mainly divided into two types (Fig. 2a). One
type included six genes without intron, including TaY-
UCCA1-A, TaYUCCA1-B, TaYUCCA1-D, TaYUCCA8-A,
TaYUCCA8-B, and TaYUCCA8-D, and the lengths of these
six genes did not differ substantially. The number of amino
acids ranged from 416 to 428 (Table S2). Another type had
an intron and two exons, including TaYUCCA8-B2, TaY-
UCCA17-A, and TaYUCCA18-B. Moreover, TaYUCCA8-
B2 and TaYUCCA8-B genes formed two transcripts by the
same gene as indicated by alternative splicing methods.
In group VI, four, five, and nine exons were observed in
different genes. The DNA sequence of TaYUCCA-5D was
longer than that of group VI (approximately 14 kb) mainly
because the first intron was larger (approximately 13 kb,
which was the largest intron of the 63 genes, Fig. 2a). In
addition to the TaYUCCA12-D2 gene, which had the sec-
ond longest DNA and highest number of introns in group
VI, TaYUCCA13-A was the smallest gene in the TaYUCCA​
family, and it contained five exons and four introns (Fig. 2a).
Among them, the introns of TaYUCCA11-A, TaYUCCA11-B,
TaYUCCA11-D, and TaYUCCA12-A2 were relatively large,
and the sequence of TaYUCCA12-A2 gene intron reached
approximately 6.5 kb. In group VI, 24 genes encoded 382
amino acids. The evolutionary relationship of these 24 genes
was close (Fig. 2a). The exon/intron structure between the
genes in the same group, especially homologous genes, was
relatively conservative, but with some exceptions, indicat-
ing their functional diversification. The pI of the 63 TaY-
UCCA proteins was predicted to be 5.8 (TaYUCCA7-B)
to 9.24 (TaYUCCA16-A2), which represent weak acid and
weak base, respectively. The MW was between 41.1788 and
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58.761 kDa. Subcellular localization showed that these 63
proteins were located in the nucleus, chloroplast, cytoplasm,
and secretory (Table S2).
Expression pattern of TaYUCCA genes in different
tissues
The publicly available RNA-Seq data set (https​://wheat​.pw.
usda.gov/Wheat​Exp/) was used to analyze the expression of
the TaYUCCA​gene under different stresses and in different
tissues of wheat, including roots, stems, leaves, and grains.
Through sequence searching, we found 22 TaYUCCA​genes
from the WheatExp database as shown in Table S3. The
analysis of the database [27] showed that seven TaYUCCA​
genes were expressed on the 12th day of kernel. Among
these genes, TaYUC10.1, TaYUC10.2, and TaYUC10.3
showed high expression levels, and the expression of these
three genes was highest in the endosperm, followed by the
inner pericarp, and the least expressed in the outer pericarp
(Fig. 3a).
We further analysed the TaYUCCA​gene expression at dif-
ferent growth stages using the database [28]. In the Wheat-
Exp data, we found that the gene was expressed in five wheat
tissues at nine different developmental stages (Fig. 3b). In
the Fig. 3b, the three alleles of TaYUCCA2-A, TaYUCCA2-B,
and TaYUCCA2-D were expressed similarly. Their expres-
sion levels were high in the roots during the three-leaf and
meiotic stages and low in the other tissues. TaYUC10.1,
TaYUC10.2, and TaYUC10.3 were highly expressed in the
medium milk of grains. While the expression levels in the
kernel watery ripe and soft dough of grains and other stages
were extremely low or even absent. The expression level of
Fig. 3  Expression of TaYUCCA​ gene at 12  days post-anthesisin dif-
ferent layers of seeds and in different tissues. a Expression of TaY-
UCCA​ gene at 12 days post-anthesisin different layers of seeds. The
RNA-sequence data was obtained from endosperm, inner and outer
seed pericarp tissues of immature seeds, three biological replicates
of each tissue were used [27]. b The expression of TaYUCCA​ genes
in different developmental stages. The RNA-sequence data were
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Molecular Biology Reports (2021) 48:1269–1279
TaYUCC9-D was relatively high in the kernel watery ripe of
grain and spikes of two nodes and anthesis, and the expres-
sion level of TaYUCC9-A was high in the root of seeding,
two nodes of spike, and stem of spike at 1 cm. The expres-
sion levels of the two genes were both high in the spikes of
the two nodes. The expression levels of TaYUCCA3-A and
TaYUCCA3-B genes were relatively high in the roots in the
three-leaf and meiotic stages. Thus, the functions of the TaY-
UCCA​gene alleles were similar, but the expression profiles
of the above alleles were slightly different at different devel-
opmental stages. In addition, TaYUCCA5-A, TaYUCCA7-D,
TaYUCCA1-A, and TaYUCCA8-B were highly expressed in
the roots at different stages, and the expression level of TaY-
UCCA5-A in the stems of the two nodes was relatively high.
TaYUCCA12-B5 and TaYUCCA9 were highly expressed in
the medium milk of grain and at different stages of stem
development. This finding may be caused by certain overlap
in the function of certain genes in the TaYUCCA​gene family
and to the evolution of the genome.
Analysis of the grain layer developmental time course in
WheatExp data [29] showed that the TaYUCCA2-A, TaY-
UCCA2-B, and TaYUCCA2-D genes were highly expressed
in the transfer cells (TCs) at 20 days post anthesis (DPA).
The expression levels of TaYUC10.1, TaYUC10.2, and
TaYUC10.3 genes were higher in the embryo at 10 DPA and
in the starch endosperm (SE) at 20 DPA. The TaYUCCA3-B
and TaYUCCA1-A genes were highly expressed in the aleu-
rone and endosperm at 30 DPA, and most of the genes were
highly expressed in TCs at 20 DPA. The TaYUCCA21-A
gene was highly expressed in the SE at only 20 DPA, and
the TaYUCCA13-D gene was only expressed in the whole
endosperm at 20 DPA (Fig. 3c).
obtained from leaf, root, stem, spike and grain of Chinese spring
[28]. c The expression of TaYUCCA​ genes in different various parts
of grains in different periods. The RNA-sequence data were obtained
from endosperm, starchy endosperm, transfer cell, aleurone, and
tarchy endosperm from wheat expression database [29]. Data down-
loaded from http://wheat​.pw.usda.gov/Wheat​Exp/. Values are in frag-
ments per kilobase of exon per million reads mapped (FPKM)
Molecular Biology Reports (2021) 48:1269–1279 1275
Under drought and heat stress, only a few TaYUCCA​
genes were upregulated than that in the control in WheatExp
data [30]. Most of the genes were downregulated or even not
expressed after stress (Fig. 4a).
Expression pattern of TaYUCCA genes in different
stresses
There are 15 TaYUCCA​genes induced by drought and heat
stress. After drought stress for 1 h (DS-1H), the expres-
sion of TaYUCCA2-D was upregulated, whereas that of
TaYUCCA2-A and TaYUCCA2-B was downregulated. The
expression of TaYUCCA3-B and TaYUCCA9-D genes after
DS-6H and that of TaYUCCA6-A gene after DS-6H and
heat stress for 1 h (HS-1H) were upregulated. The expres-
sion level of TaYUCCA10.1 gene was upregulated after
HS-1H, whereas that of TaYUCCA10.2 and TaYUCCA10.3
genes was downregulated under heat stress and the oth-
ers 9 TaYUCCA​ genes were downregulated induced by
drought and heat stress (Fig. 4a). By qRT-PCR, we further
analyzed seven TaYUCCA​ genes response to drought and
heat stress. Among these seven genes, the expression lev-
els of TaYUCCA1-A (Fig. 4b), TaYUCCA2-B (Fig. 4c) and
TaYUCCA3-A (Fig. 4d) were affected by DS and DH-1H,
the result were opposite with the WheatExp database
(Fig. 4a). The TaYUCCA6-A (Fig. 4e) was downregulated
by HS-1H and different from the WheatExp database,
Fig. 4  TaYUCCA​ gene expression during drought or heat stress.
a TaYUCCA​ gene expression during drought, heat stress or their
co-stress, data downloaded from http://wheat​.pw.usda.gov/Wheat​
Exp/ [30]. Values are in reads per kilobase of exon per million reads
mapped (RPKM). b–h qRT-qPCR assessment of the expression of
selected wheat TaAP genes induced by drought, heat stress or their
whereas the TaYUCCA8-B (Fig. 4f) and TaYUCCA10.2
(Fig. 4g) were downregulated under drought, heat stress
and their co-stress, which were consistent with the results
of the WheatExp database (Fig. 4a). The TaYUCCA10.1
gene responsed to drought, heat stress and their co-stress,
especially under treatment for HS-1H, where its expres-
sion was the highest and was 31 times of that of the con-
trol, whereas in the WheatExp database TaYUCCA10.1
only had a high expression in HS-1H (Fig. 4a, h). These
quantitative results were slightly different to those at the
WheatExp database, possibly because of the differences
between individuals.
In this study, we analyzed whether TaYUCCA​ family
genes were involved in the interaction with powdery mil-
dew by obtaining leaf samples of wheat cultivar Chinese
Spring induced with powdery mildew for 0, 1, 2, 3, 4 and
5  days for the transcriptome analysis [24]. The analy-
sis of the transcriptome of Chinese Spring showed that
TaYUCCA7-A, TaYUCCA17-A, and TaYUCCA18-B were
upregulated expression genes during the powdery mildew
induction time series (Fig. 5a). We selected two genes for
further quantitative analysis, and found that TaYUCCA7-A
was significantly induced by powdery mildew, especially
on the 3rd day (Fig. 5b), while TaYUCCA18-B has a stable
expression under induction of powdery mildew (Fig. 5c).
This finding indicated that the TaYUCCA​genes might have
diverse functions when participating in the interaction
with powdery mildew.
co-stress at different times. Relative expression levels of TaYUCCA1-
A (b), TaYUCCA2-B (c), TaYUCCA3-A (d), TaYUCCA6-A (e), TaY-
UCCA18-B (f) TaYUCC10.2 (g) and TaYUCCA10.1 (h). Different
letters above the bars indicated statistically significant differences
(P < 0.05) as obtained by one-way ANOVA (LSD-Duncan method).
DS drought stress, HS heat stress, HD their co-stress
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1276
Fig. 5  TaYUCCA​ genes that are upregulated expressed in time series
induced by powdery mildew. a Heat map showing the expression pro-
files of three TaYUCCA​ genes that are upregulated expressed in time
series induced by powdery mildew. RNA-seq data was obtained from
leaves induced by powdery mildew in Chinese Spring. Correspond-
ing transcripts per 10 million values were used to construct heat maps
and scale indicators for expression of genes. b–c qRT-PCR assess-
Discussion
The YUCCA​gene has been widely studied, and 11 YUCCA​
genes of A. thaliana were strictly regulated during A. thali-
ana growth and development [18]. Yamamoto et al. reported
that OsYUC1 was expressed in almost all organs tested
and participates in rice growth and function [21]. Studies
on YUCCA​ genes in wheat are limited. Li et al. obtained
three genes of TaYUCCA10 by cloning and found that TaY-
UCCA10 might play an important role in seed develop-
ment [31]. Yang et al. found that specific-expressing TaY-
UCCA10.3 in young seeds of transgenic wheat Bobwhite
could increase the auxin and the protein content of the seeds
significantly and speculated the TaYUC10.3 had an impor-
tant role in IAA- ZT biosynthesis and degradation in wheat
[32]. In this study, we identified 63 wheat YUCCA​genes and
found no gene distribution on the homologous chromosome
group no. 6. Exactly 24, 19, and 20 gene loci were distrib-
uted in A, B, and D groups, respectively (Table S2).
Comparison of the genomes of one organisms is a rapid
method to understand the genomic structure and function of
another unknown organism’s genome. Studies about YUCCA​
gene in A. thaliana and rice have been developed, and their
genomes of these species can be used as a reference to infer
the function of their homologous genes in wheat. By con-
structing the phylogenetic tree, we found that TaYUCCA​,
AtYUCCA​, and OsYUCCA​genes have a certain relationship
in evolution (Fig. 2b). As shown in Fig. 2b, wheat, rice,
and A. thaliana clustered in the group III, IV, and V, while
wheat and rice clustered in the group I and II, and wheat
and A. thaliana clustered in the group VI. The YUCCA​
genes had different functions, such as the OsYUCCA5 was
auxin-related gene and induced expression by alginate
13
Molecular Biology Reports (2021) 48:1269–1279
ment of the expression of selected wheat TaYUCCA​genes induced by
powdery mildew at different times. Relative expression levels of TaY-
UCCA7-A (b) and TaYUCCA18-B (c). Different letters above the bars
indicated statistically significant differences (P < 0.05) as obtained by
one-way ANOVA (LSD-Duncan method). The TaActin was used as
the internal control to normalize the real-time PCR data. Error bars
indicates standards errors from three biological repetitions
oligosaccharides (AOS) in rice tissues to accelerate auxin
biosynthesis and transport, and reduced indole-3-acetic
acid (IAA) oxidase activity in rice roots [33], the OsCOW1
was required for maintaining water homeostasis and an
appropriate root-to-shoot ratio in group II [34]. Yamamoto
et al. found the overexpression of OsYUCCA1 inhibited
leaf growth and root elongation, and which encodes a key
enzyme contributing to IAA biosynthesis, was expressed
in almost all of the organs tested, and functions in rice
growth and development [21]. AtYUCCA1, 2, 4, and 6 were
expressed mainly in the inflorescence apex and flowers, and
were found to be important in flower development based
on yucca1/2/4/6 quadruple-mutant analysis [18]. Kim et al.
found the overexpression of AtYUCCA6 and AtYUCCA7
enhanced drought resistance in tomato and A. thaliana,
respectively [35]. Fujino et al. found that OsYUCCA1, 3,
6, and 7 played roles in embryonic, shoot, root, and panicle
development [36]. Cheng et al. found that the expression
patterns of AtYUCCA10 and AtYUCCA11 overlap with those
of AtYUCCA1 and AtYUCCA4 during embryogenesis, and
both AtYUCCA1 and AtYUCCA4 were expressed in discrete
groups of cells throughout embryogenesis [19]. Li et al.
studied the TaYUCCA10 genes found that this gene might
involve in morphogenesis [31]. These phenomena indi-
cated that YUCCA​genes function were diverse, but YUCCA​
genes could play an important role in the wheat growth and
development.
Gene duplication plays an important role in the evolution
of various organisms [37]. For most gene families, tandem
repeats result in new functions for repetitive genes [38]. In
this study, we found that a part of the TaYUCCA​ gene is
involved in tandem repeats in the wheat genome (Fig. 1).
The exon/intron diversity of gene family members plays an
Molecular Biology Reports (2021) 48:1269–1279 1277
important role in evolution, and the multigene family works
via three main mechanisms, namely, exon/intron gain/loss,
exonization/pseudo exonization, and insertion/deletion [39].
Based on multiple sequence alignments, the amino acid
sequences of the genes clustered in the same group were
highly similar, and the exon/intron structures of genes in
the same group were mostly identical (Fig. 2a). Some genes
such as TaYUCCA9-A, and TaYUCCA9-B differed in terms
of structure and sequence length (Fig. 2a). However, the
similarity of the encoded amino acid sequences was very
high, indicating the convergence of alleles and the conserva-
tive structure of the TaYUCCA​gene.
By searching the WheatExp database, we further
understood the function of the TaYUCCA​ gene. The
TaYUCCA​ gene family had diverse functions especially
in group VI. For instance, TaYUCCA19-A was detected
in the whole endosperm in 20 DPA and transfer cells in
20 DPA; TaYUCCA21-A gene had high expression level
of starchy endosperm in 20 DPA; TaYUCCA12-B5 was
abundantly expressed in the kernel watery ripe of grain
period; TaYUCCA12-B, TaYUCCA12-B2, TaYUCCA12-
B3, TaYUCCA12-B4, TaYUCCA1-B, TaYUCCA20-B, and
TaYUCCA20-B2 were not detected in different stages of
wheat development; TaYUCCA13-D3 was expressed in the
spike of meiosis and anthesis; TaYUCCA13-D was abun-
dantly expressed in the kernel watery ripe of grain period;
and TaYUCCA13-D, TaYUCCA15-D and TaYUCCA16-D
were not detected in the different stages of wheat develop-
ment (Fig. 3b, c). These expression levels of the TaYUCCA​
gene involved in the tandem repeat region slightly differed
under the different growth stages of wheat, suggesting that
the function of the gene may be altered by replication, and
tandem repeats result in the silencing of gene expression
after duplication. Hence, tandem repeats will change the
regulation mechanism of genes. In addition, the expression
of alleles in different tissues at different stages was similar.
For example, the TaYUCCA2-A, TaYUCCA2-B, and TaY-
UCCA2-D genes were abundantly expressed in the roots
of the three-leaf and meiotic stages (Fig. 3b) and in TCs
at 20 DPA (Fig. 3c), indicating that the alleles may exhibit
similar functions. Moreover, the TaYUC10.1, TaYUC10.2,
and TaYUC10.3 genes were abundantly expressed in the
endosperm (Fig. 3a), medium milk (Fig. 3b), and whole
endosperm at 10 DPA and starchy endosperm at 20 DPA
(Fig. 3c). The TaYUC10.1, TaYUC10.2 and TaYUC10.3
gene were highly expressed during the seed develop-
ment period [31, 32]. Considering that only some gene
expression levels can be found in the expression database,
the expression of other genes remains unclear. Although
the amino acid sequences among the alleles were highly
similar, differences in individual loci were still observed.
These differences might cause changes in gene function,
and these gene functions should be further studied. In
addition, some genes such as TaYUCCA7-D, TaYUCCA1-
A, and TaYUCCA8-B, which were highly expressed in
roots at different stages (Fig. 3b), showed distant homolo-
gous relationships but similar expression patterns.
Subsequently, we found that some TaYUCCA​ family
genes were involved in drought and heat stress such as TaY-
UCCA1-A, TaYUCCA2-B, TaYUCCA3-A, TaYUCCA10.1,
TaYUCCA8-B and TaYUCCA10.2 (Fig. 4). Figure 2a showed
that the six genes above belongs to groups IV, V, and VI.
Wang et al. found that the increase in the expression of YUC​
gene in PEG-treated plants resulted in auxin synthesis and
transport regulation in drought stress [40]. By analyzing the
transcriptome induced by powdery mildew in wheat Chinese
Spring, we found that most of TaYUCCA​ genes were not
induced by powdery mildew fungus, while TaYUCCA7-A
was responsive to powdery mildew. This finding might pro-
vide an important basis for future research on TaYUCCA​
genes involved in resistance to powdery mildew (Fig. 5).
Auxin is an important plant growth hormone that is syn-
thesized in many tissues of plants and may travel long dis-
tances from one cell to another through cell membranes [41].
The YUCCA​ gene is a rate-limiting step in the tryptophan-
dependent pathway for auxin synthesis, and auxin plays an
important regulatory role in plant growth and development
[10]. The YUCCA​ genes of A. thaliana and rice have been
cloned and studied [18, 19, 21, 42]. However, considering
the complexity of wheat genome composition, the study
of TaYUCCA​ gene’s function in wheat remains unclear. Li
et al. cloned a TaYUCCA10 gene and found it was highly
expressed during seed development, indicating that TaY-
UCCA10 might be involved in seed morphogenesis [31]. The
hypocotyl of A. thaliana with overexpressed TaYUCCA10.3
gene was longer, the petiole was obviously elongated, and
the leaf length was long and narrow compared with those of
wide type plants [31]. Overexpression of TaYUCCA10.3 in
wheat young seeds could increase the content of auxin and
seed protein [32]. Therefore, understanding the TaYUCCA​
gene family will enhance our researching of the function of
the TaYUCCA​genes.
Supplementary Information  The online version of this article (https​://
doi.org/10.1007/s1103​3-021-06197​-0) contains supplementary mate-
rial, which is available to authorized users.
Acknowledgements  We would like to thank the anonymous review-
ers for their suggestions and comments. All authors thank the raw
data, which can be downloaded from the public database. The high
credibility protein sequences of wheat (iwgsc_refseqv1.0_High-
Conf_PROTEIN_2017Mar13). The wheat YUCCA​ gene and protein
sequences were obtained from the IWGSC RefSeq v1.0 assembly
(https​://wheat​-urgi.versa​illes​.inra.fr/Seq-Repos​itory​/Assem​blies​).
The expression pattern of the TaYUCCA​ genes on different stresses
and different tissues of wheat including roots, stems, leaves, skips,
and grains was from the RNA-Seq data set (https:​ //wheat.​ pw.usda.gov/
Wheat​Exp/) [27-30].
13

1278
Author contributions  DF, HW conceived the study. YY and TX per-
formed the experiments. YY and TX carried out the analysis. YY and
DF designed the experiments. YY and DF wrote the manuscript. All
authors read and approved the final manuscript.
Funding  This work was supported by the Grants from the National
Natural Science Foundation of China (31771777); Shangdong “Double
Tops” Program; and the Overseas Visiting Programme for Gradate
Mentors of Shandong Province.
Compliance with ethical standards  looking beyond detox. Trends Plant Sci 12:412–418
Conflict of interest  The authors declare that they have no conflict of
interest to report.
Research involving human and animal rights  This article does not
contain any studies with human participants or animals performed by
any of the authors.
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