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Characterization and expression analysis of cytokinin biosynthesis genes in

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Plant Growth Regul (2017) 82:139–149
DOI 10.1007/s10725-016-0246-z
ORIGINAL PAPER
Characterization and expression analysis of cytokinin biosynthesis
genes in Fragaria vesca
Xianna Mi1 · Xiaojing Wang1 · Han Wu1 · Lijun Gan2 · Jing Ding1 · Yi Li1,3
Received: 8 September 2016 / Accepted: 30 December 2016 / Published online: 13 January 2017
© Springer Science+Business Media Dordrecht 2017
Abstract  Strawberry is one of the most economically
important fruit crops in the world. Cytokinins (CKs) play
a critical role in plant growth and development, as well as
the stress response, and the level of CKs in plants is regu-
lated by synthesis and degradation pathways. The key syn-
thetic enzymes of CKs are isopentenyl transferases (IPTs)
and LONELY GUYS (LOGs). We surveyed the straw-
berry genome and identified seven FvIPT genes and nine
FvLOG genes. We analyzed gene structures, conserved
domains, and their phylogenetic relationships with rice
and Arabidopsis. The isoelectric points and glycosylation
sites of the proteins were predicted. We also analyzed tis-
sue- or organ-specific expression patterns of the FvIPT and
FvLOG genes. The FvIPT and FvLOG genes showed dif-
ferent expression profiles in different organs. Most FvIPT
and FvLOG genes were down-regulated in response to
osmotic stress, high-temperature treatment, and exogenous
abscisic acid (ABA) application, suggesting possible roles
of these genes in the plants’ resistance to abiotic stresses.
Electronic supplementary material  The online version of this
article (doi:10.1007/s10725-016-0246-z) contains supplementary
material, which is available to authorized users.
* Lijun Gan
ganlj@njau.edu.cn
* Jing Ding
jding@njau.edu.cn
1 and dihydrozeatin (DZ) (Sakakibara 2006). Active isopre-
State Key Laboratory of Crop Genetics and Germplasm
Enhancement and College of Horticulture, Nanjing
Agricultural University, Nanjing, People’s Republic of China
2
College of Life Sciences, Nanjing Agricultural University,
Nanjing, People’s Republic of China
3
Department of Plant Science and Landscape Architecture,
University of Connecticut, Storrs, CT 026269, USA
 
In addition, we found that the results of bioinformatics
analyses to identify cis-regulatory elements may not be
consistent with experimental expression data; thus, com-
puter-predicted putative cis-elements need to be confirmed
by experiments. Our systematic analyses of the FvIPT and
FvLOG families provide a foundation for characterizing the
function of these genes in the regulation of growth, devel-
opment, and stress tolerance in Fragaria vesca, as well as
a reference for improving stress tolerance by manipulating
CK content.
Keywords  Strawberry · Cytokinin · FvIPT · FvLOG ·
Abiotic stress
Introduction
Cytokinins (CKs) are a class of adenine-derived plant hor-
mones that play a crucial role in multiple processes related
to growth and development, such as seed development,
shoot initiation, and leaf senescence (Chandler and Werr
2015; Schaller et al. 2015). Moreover, evidence shows that
CKs are also involved in stress responses (Hare et al. 1997;
Nishiyama et al. 2011).
Natural cytokinins possess either an isoprenoid or an
aromatic side chain at the ­N6 position. Common natural
isoprenoid cytokinins in higher plants primarily consist of
isopentenyl adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ),
noid cytokinins include iP, tZ, and cZ, whereas cytokinin
ribosides are common transport forms with no or very
weak cytokinin activities (Lomin et al. 2015). Over the past
decade, cytokinin biosynthesis pathways have been ana-
lyzed and well characterized in rice and Arabidopsis. The
key enzymes involved in CK biosynthesis are isopentenyl
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140
transferases (IPTs) and LONELY GUYs (LOGs) (Kam-
ada-Nobusada and Sakakibara 2009; Kieber and Schaller
2014). The IPT and LOG gene families are expressed in
nearly all tissues of rice and Arabidopsis (Kuroha et  al.
2009; Miyawaki et al. 2004; Takei et al. 2004).
The IPTs, which are the central rate-limiting enzymes of
CK biosynthesis, consist of adenylate-IPTs and tRNA-IPTs
(Sakakibara 2006). The initial step in cytokinin biosyn-
thesis is catalyzed by adenylate-IPTs to form iP ribotides.
These iP nucleotides can be converted to the correspond-
ing tZ nucleotides by cytochrome P450 monooxygenases
CYP735As (Takei et al. 2004). In higher plants, iP and tZ
nucleotides are precursors for most iP and tZ-type cyto-
kinins. The biosynthesis of cZ is initiated by tRNA-IPTs,
which catalyze the isopentylation of tRNA using dimethy-
lallyl diphosphate (DMAPP) (Sakakibara 2006; Kudo et al.
2010). The common feature of the two classes of IPTs is
a conserved IPPT-binding domain. In Arabidopsis plants,
nine IPT genes (designated as AtIPT1–AtIPT9) have been
identified, seven of which (AtIPT1 and AtIPT3–AtIPT8)
are adenylate-IPTs, and two genes are transfer RNA-
IPTs (AtIPT2 and AtIPT9) (Takei et  al. 2001). In the rice
genome, 10 IPT genes (OsIPT1 to OsIPT10) have been
identified, and OsIPT9 and OsIPT10 were found to be
involved in tRNA prenylation (Sakamoto et  al. 2006).
Cytokinin biosynthesis genes were also identified in the
Rosaceae family. For example, seven PpIPT genes were
identified in peach (Prunus persica) (Immanen et al. 2013),
and MdIPT3a was cloned from apple (Malus pumila Mill.)
(Zhu et  al. 2012). Seven FvIPTs and nine FvLOGs were
identified in Fragaria vesca (Kang et al. 2013).
CK-ribotides are precursors that are converted into bio-
logically active cytokinins. It was found that 5′-monophos-
phate phosphoribohydrolase, designated LONELY GUY, is
a novel cytokinin-activating enzyme that directly converts
inactive CK-ribotides to the free-base forms. LOG was
first discovered in rice by a genetic screen for defects in the
maintenance of shoot meristems (Kurakawa et al. 2007). In
Arabidopsis, nine LOG genes (AtLOG1 to AtLOG9) were
predicted as homologs of rice LOG. Among them, seven
proteins (AtLOG1 to AtLOG5, AtLOG7, and AtLOG8)
were localized in the cytosol and in nuclei that showed
activity of LOG enzymes (Kuroha et al. 2009). All LOGs
are characterized by the presence of a conserved lysine
decarboxylase-binding domain within their proteins.
CK biosynthesis and homeostasis are finely controlled
by biosynthetic and metabolic enzymes (Frébort et  al.
2011). Cytokinin dehydrogenases/oxidases (CKXs) are
responsible for irreversible degradation of CKs (Galuszka
et  al. 2007). When environmental changes such as
increased drought, soil salinity, and/or temperature occur
during the development of plants, cytokinin biosynthesis
and metabolic genes respond to the stress (Ha et al. 2012).
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Plant Growth Regul (2017) 82:139–149
Under salt- and osmotic-stress conditions, the expression
of most examined Arabidopsis IPT genes was repressed in
2-week-old seedlings. The expression of AtIPT1, AtIPT3,
and AtIPT7 was reduced during dehydration treatment,
whereas the expression of AtIPT5 was slightly increased
after 10  h of dehydration. Under salt stress, the expres-
sion of AtIPT1 and AtIPT3 was suppressed in Arabidopsis
seedlings (Nishiyama et al. 2011). The IPT genes in maize
and tobacco responded to drought and salt conditions in
a manner similar to Arabidopsis (Havlová et  al. 2008;
Vyroubalova et  al. 2009). Additionally, after exogenous
ABA treatment, the expression of IPT genes was altered in
Arabidopsis seedlings (Nishiyama et al. 2011). The expres-
sion of OsIPT2, OsIPT4, and OsIPT7 was down-regulated
in rice after treatment with ABA for 2 h (Tsai et al. 2012).
Currently, there are very few reports on the expression of
LOG genes under stress conditions, but research on LOG
from Arabidopsis multiple mutants suggests that the LOG-
dependent pathway is the main route of cytokinin activa-
tion (Tokunaga et al. 2012). The results from studies on CK
biosynthesis genes in rice and Arabidopsis provide a refer-
ence for functional characterization of their homolog genes
in other plants.
Strawberry is an important horticultural crop world-
wide (Pinto et al. 2010; Zhang et al. 2008). Because of its
shallow root distribution, diminutive stature, and short life
cycle, a number of environmental stresses adversely affect
strawberry growth and productivity (Shulaev et  al. 2011).
The availability of whole-genome sequencing information
for F. vesca enables us to conduct genome-wide identifica-
tion and analysis of the IPT and LOG gene families (Shu-
laev et al. 2011). Taking into account the importance of CK
biosynthesis genes in the regulation of stress tolerance, we
identified and characterized the expression profiles of IPT
genes and LOG genes in seedlings of the diploid woodland
strawberry (F. vesca) model species, and analyzed their
expression patterns under different stress conditions. The
information gained from these novel studies will provide
insight into the functions of CK biosynthesis genes and
help identify candidate genes for breeding to enhance stress
resistance in F. vesca.
Materials and methods
Identification of FvIPT and FvLOG gene families
in woodland strawberry
For identification of FvIPT and FvLOG genes in woodland
strawberry, we retrieved the F. vesca genome and proteome
sequences from the phytozome website (http://www.phyto-
zome.net/strawberry.php/). Local BLAST (P value = 0.001)
search of all the CDS in woodland strawberry proteome
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Plant Growth Regul (2017) 82:139–149 141
was carried out by using the HMM profile of the IPT and
LOG domain as query. The HMM profiles of IPT domain
(ID:PF01715) and LOG domain (ID:PF03641) were down-
loaded from the Pfam database (http://pfam.Xfam.org/).
The FvIPT and FvLOG proteins deduced from DNA
sequences that contain start and stop codons, were selected
for further analysis. Selected proteins were searched against
the PFAM database to confirm the presence of domain sig-
natures. Structures of FvIPT and FvLOG genes were deter-
mined by the GSDS tool (http://gsds.cbi.pku.edu.cn/).
Physiological and biochemical property analysis
of FvIPT and FvLOG proteins
Multiple alignments of amino acid sequences were per-
formed with MEGA v5.10 software (http://www.megasoft-
ware.net/). The resulting amino acid sequence alignments
were then used to guide the alignments of domains. Phylo-
genetic trees were constructed by the bootstrap neighbor-
joining (NJ) method with a Kimura 2-parameter model by
MEGA. The stability of internal nodes was assessed by
bootstrap analysis with 1000 replicates.
The isoelectric points (pI) of the FvIPT and FvLOG
proteins were predicted using Compute pI/Mw software
(http://www.expasy.ch/tools/pi_tool.html/). The glycosyla-
tion sites were predicted using NetNGly (http://www.cbs.
dtu.dk/services/NetNGlyc/).
Cis‑element analysis of the putative promoter regions
of FvIPT and FvLOG genes
To understand transcriptional regulation and the potential
expression patterns of the FvIPT and FvLOG genes, the
1000  bp sequences up-stream of the transcriptional start
site of each FvIPT and FvLOG gene were chosen. These
sequences were used to query the PlantCARE database
(http://bioinformatics.psb.ugent.be/webtools/plantcare/
html/), and the putative cis-regulatory elements and func-
tions of the promoter sequences were identified.
Plant growth and treatments
The plant materials (F. vesca cv ‘Hawaii 4’) were grown
in the soil at the plant growth room in Nanjing Agricul-
tural University. The roots, stems, stolons, leaves, flowers
(pre-fertilization), young fruits (2–4  days after anthesis)
and big fruits (10–13 days after anthesis) were sampled to
analyze the tissue- or organ-specific expression of the dif-
ferent genes. All the materials were sampled from at least
five plants.
For gene expression analysis, strawberry seedlings
grown in 1/5 Hoagland solution for 3  weeks with the fol-
lowing settings: 60% relative humidity, 27 °C and a light
photoperiod of 16 h light/8 h dark. The 1/5 Hoagland solu-
tion supplemented with 25% polyethylene glycol (PEG)
6000 was used for osmotic stress treatment. Leaves and
roots were collected at 0, 3 and 8  h of the osmotic treat-
ment. Heat shock was applied by raising the temperature
in the culture chamber to 38 °C. The leaves and roots
were sampled at 0, 3 and 8  h after treatments. For ABA
treatment, the 3-week-old seedlings were grown in 1/5
Hoagland solution with the addition of 100  μM ABA.
Leaves and roots were sampled at 0, 0.5, and 1 h after treat-
ment. The materials were collected and quickly frozen in
liquid nitrogen and stored at −80 °C until needed.
RNA extraction and qRT‑PCR analysis
The total RNA was extracted from samples using the 2%
CTAB and then reverse transcribed into cDNA using the
PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China).
The gDNA eraser in the kits was used to eliminate DNA
to prevent DNA contamination. The primers used for qRT-
PCR were designed using the website (http://www.idtdna.
com/site) and the Primer (version 6.0) software and verified
for amplification efficiency. Primers are listed in Table S1.
We had three bio-replicates and three tech-replicates in
these experiments. Amil-Ruiz et al. (2013) tested 13 straw-
berry genes in different cultivars, tissues, biotic stresses
and developmental stages such as ripening and senescent
stages and evaluated their suitability as reference genes.
As a result, the authors have recommended FaGAPDH2 as
one of reference genes for relative quantification of gene
expression in strawberry. Therefore, we used the GAPDH2
gene as a reference gene. The volume of each qRT-PCR
reaction is 20 μL, which contained 1 μL of cDNA, 10 μL
SYBR Green Master Mix Reagent (Takara, Japan) and
2 μL specific primers. The qRT–PCR was performed using
MyiQ Single color Real-Time PCR Detection System
(Bio-rad, Hercules, CA, USA) with SYBR Premix Ex-Taq
(TaKaRa, Dalian, China). The PCR conditions were: 95 °C
for 30 s; followed 40 cycles of 95 °C for 5 s and 60 °C for
30 s; and at the end, 65 °C for 15 s.
The relative expression levels were normalized to the
quantification of GAPDH2 using the 2− ΔΔCT method (Livak
and Schmittgen 2001).
Results
Identification and classification of IPT and LOG genes
in strawberry
Identification of FvIPT and FvLOG genes was completed
using local BLAST (P value = 0.001) and the HMM pro-
file of the IPT and LOG domains as a query. Seven FvIPT
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142 Plant Growth Regul (2017) 82:139–149

and nine FvLOG genes that contained conserved domains
were obtained from the woodland strawberry genomes. The
FvIPT and FvLOG genes were named according to previ-
ous reports (Kang et  al. 2013). Sequence features of the
FvIPTs and FvLOGs are summarized in Table 1.
The seven FvIPT genes are distributed unevenly as fol-
lows on three chromosomes: four genes are located on
chromosome III, two on chromosome IV, and only one
FvIPT gene is found on chromosome II (Table 1). Next, we
analyzed the tandem duplications of the FvIPT gene fam-
ily. However, no tandem duplicate gene pairs were found
among FvIPT genes. The FvIPT1, FvIPT3, FvIPT4, and
FvIPT5 genes do not have any introns, whereas FvIPT6
has 2 introns, and FvIPT2 and FvIPT7 contain 15 and 16
introns, respectively (Table  1; Fig.  1a). This feature was
also observed in genes encoding IPTs from Arabidopsis as
the tRNA-dependent isopentenyl transferase group; AtIPT2
and AtIPT9 contain 10 and 11 exons, respectively (Fig.
S1a). We predicted that FvIPT2 and FvIPT7 are tRNA-IPT
genes that may be involved in tRNA prenylation. Addi-
tionally, we identified the glycosylation sites in FvIPT2,
FvIPT5, FvIPT6, and FvIPT7 using the tool NetOGlyc
(http://www.cbs.dtu.dk/services/NetOGlyc/) (Table 1). Gly-
cosylation plays an important role in the regulation of pro-
tein folding, biological activity and protein stability (Olc-
zak et al. 2003; Tran et al. 2010). This result indicates that
FvIPT2, FvIPT5, FvIPT6, and FvIPT7 may have a complex
regulation patterns in strawberry.
The nine FvLOG genes are also distributed on chro-
mosomes II, III, and VI. Five of the nine genes (FvLOG1,
FvLOG2, FvLOG7, FvLOG8, and FvLOG9) are distributed
on chromosome III, and the other four genes are evenly dis-
tributed on chromosomes II and VI (Table 1). No tandem
duplications were found among the FvLOG genes. The
CDS lengths of the FvLOG genes (612–921) are shorter
than those of the FvIPT genes (882–2427), but all FvLOG
genes have six to eight introns (Table 1; Fig. 1b). FvLOG5,
FvLOG6, FvLOG7, and FvLOG9 each have one predicted
glycosylation site, and the remaining FvLOG genes do not
contain glycosylation sites (Table 1).
Phylogenetic analyses of FvIPT and FvLOG families
To examine the phylogenetic relationships of the IPT and
LOG gene families from the woodland strawberry, Arabi-
dopsis, and rice, we performed phylogenetic analyses using
the neighbor-joining method in MEGA5.10 software. The
structure and domains of IPT and LOG gene families are
relatively conserved among the three plant species (Fig. 2).
Six IPT proteins in strawberry have only one IPPT
(IPP transferase) domain, and FvIPT7 contains two addi-
tional domains (Fig. 2a). The FvIPT2, AtIPT2, and OsIPT9
genes belong to the same clade, and FvIPT7, AtIPT9, and
OsIPT10 belong to another clade. These two branches
are supported by high bootstrap values (Fig.  2a). Based
on phylogenic analysis, the FvIPT2, AtIPT2, and OsIPT9
genes constitute the oldest branch on the tree and are plant
tRNA-IPT genes of eukaryotic origin. In contrast, FvIPT7,
AtIPT9, and OsIPT10 belong to another clade and are
plant tRNA-IPT genes of prokaryotic origin (Fig. 2a). The

Table 1  General information of FvIPT and FvLOG genes in strawberry

Gene Gene no. Chromosomal Full CDS (bp) Genomic (bp) No. of intron pI Mw (kDa) N-gly-
cosyla-
tion

FvIPT1 gene03725 IV 1044 1044 0 7.06 38.93 0

FvIPT2 gene23509 III 2154 8774 15 5.36 80.22 6
FvIPT3 gene30601 III 882 882 0 5.67 33.01 0
FvIPT4 gene27343 III 915 915 0 4.89 34.13 0
FvIPT5 gene27842 II 990 990 0 5.71 37.08 1
FvIPT6 gene06080 IV 1191 3242 2 6.21 44.25 2
FvIPT7 gene27180 III 2427 9076 16 8.22 88.86 3
FvLOG1 gene16128 III 651 2821 6 5.56 23.82 0
FvLOG2 gene30641 III 651 2342 6 5.53 23.69 0
FvLOG3 gene09744 VI 711 3568 7 5.81 25.84 0
FvLOG4 gene15306 VI 648 4003 6 5.1 23.63 0
FvLOG5 gene08500 II 627 1788 6 6.38 22.76 1
FvLOG6 gene02714 II 789 3182 7 6.00 28.28 1
FvLOG7 gene30448 III 612 1503 6 5.68 21.87 1
FvLOG8 gene30673 III 708 1451 6 4.87 25.38 0
FvLOG9 gene30477 III 921 2521 8 6.42 34.49 1

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Plant Growth Regul (2017) 82:139–149 143

Fig. 1  Gene structure analysis of the FvIPT (a) and FvLOG (b) genes. Exons are shown as green boxes and introns as grey lines

Fig. 2  Phylogenetic relationships and conserved domains analysis of FvIPTs (a) and FvLOG (b) (triangle), together with their Arabidopsis
(square) and rice (circle) counterparts

tRNA-IPT genes are relatively well conserved among straw- related than those of rice are (Fig.  2a). This may suggest
berry, Arabidopsis, and rice. The ADP/ATP-dependent that they share a common ancestor of IPTs predating the
IPT genes of strawberry and Arabidopsis are more closely separation of monocots and dicots. The expansion of

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144 Plant Growth Regul (2017) 82:139–149

ADP/ATP-dependent IPT genes may have occurred after
the divergence of monocot and dicot plants. tRNA-IPT
genes catalyze the prenylation of tRNA to cZ-type prod-
ucts (CKs), and cZ binds rather weakly to Arabidopsis CK
receptors (Lomin et  al. 2015); therefore, we surveyed the
expression profiles of the other 5 FvIPT genes (FvIPT1and
FvIPT3–6), which are involved in the synthesis of bioactive
tZ- and iP-type CKs (see below).
We observed similarity to the FvIPT patterns among
genes coding for LOG proteins in strawberry, Arabidopsis,
and rice species (Fig. 2b). All LOG proteins in these three
species have highly conserved lysine-decarboxy domains.
The phylogenic tree showed that the LOGs of strawberry
and Arabidopsis were clustered together, separately from
the rice LOGs (Fig.  2b). This indicates that the LOGs
evolved separately after the monocot and dicot divergence.
Stress‑responsive cis‑regulatory elements
in the promoter regions of FvIPT and FvLOG genes
Cis-regulatory elements are located in regions upstream
of genes. They are binding sites for transcription factors,
which play major roles in abiotic stress and phytohormone
responses. A number of hormone-responsive and stress-
responsive cis-elements were found in the promoters of
FvIPT and FvLOG genes (Table 2). No cis-elements were
found in any promoter regions.
Abscisic acid (ABA)-responsive element was found in
FvIPT4 and in four LOG members (FvLOG2, FvLOG3,
FvLOG5, and FvLOG8), suggesting possible roles of
these genes in ABA signaling. Two FvIPT genes (FvIPT1
and FvIPT4) and six FvLOG genes (FvLOG1, FvLOG2,
FvLOG4, FvLOG5, FvLOG7, and FvLOG8) contain auxin-
responsive cis-elements and may function in the auxin
response. Heat stress-responsive cis-elements were found in
four FvIPT genes (FvIPT1, FvIPT3, FvIPT4, and FvIPT5)
and five FvLOG genes (FvLOG1, FvLOG2, FvLOG7,
FvLOG8, and FvLOG9), indicating possible roles of these
genes in plants’ resistance to heat stress. However, the
roles of the cytokinin-related biosynthesis genes treated by
hormones or under abiotic stress condition require further
investigation to be determined with certainty.
Tissue or organ‑specific expression of FvIPT
and FvLOG genes
Expression analysis was performed by qRT-PCR using
specific primers with equal amounts of cDNA templates
prepared from the mRNA of roots, stems, stolons, leaves,
flowers, young fruits, and big fruits of strawberry. Not all
FvIPT genes and FvLOG genes were expressed in each of
the organs, a pattern that was also observed in Arabidopsis
(Miyawaki et al. 2004). The expression of FvIPT1, FvIPT3,
FvIPT4, FvLOG3, FvLOG4, FvLOG7, and FvLOG8 was
very low in each organ, and did not show a substantial
response to any of the treatments applied; hence, we have
not presented their data. These genes are similar to AtIPT4,
AtIPT6, and AtIPT8, whose expression is undetectable in
leaves and roots (Miyawaki et al. 2006). The expression of
FvIPT5 and FvIPT6 in vegetative tissues was higher than
that in the reproductive tissues; in particular, FvIPT6 was
expressed at high levels in roots and stolons (Fig. 3a).
The FvLOG genes exhibited different expression pat-
terns among the examined tissues (Fig. 3b). FvLOG6 was

Table 2  Conserved cis-elements analysis of the FvIPT and FvLOG genes promoters

Cis-element IPT genes LOG genes
IPT1 IPT3 IPT4 IPT5 IPT6 LOG1 LOG2 LOG3 LOG4 LOG5 LOG6 LOG7 LOG8 LOG9

MeJA + + + + +
Auxin + + + + + + + +
SA + + + + + +
ABA + + + + +
GA + + + + + + + + +
Heat stress + + + + + + + + +
Low-temperature + +
Drought +
Wound + + +

+ Indicates the presence of cis-element(s)

MeJA (methyl jasmonate) responsive element: TGACG-motif, CGTCA-motif; auxin-responsive element: TGA-element, AuxRR-core; SA (sali-
cylic acid) responsive element: TCA-element; ABA (abscisic acid) responsive element: ABRE; GA (gibberellins) responsive element: GARE-
motif, P-box, TATC-box
HSE heat stress responsive element, LTR low-temperature responsive element, MBS drought-inducibility element, TC-rich repeats wound stress
responsive element

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Plant Growth Regul (2017) 82:139–149 145

Fig. 3  Relative expression profiles of FvPTs (a) and FvLOGs (b) in sis). Expression data were normalized by that of the reference gene,
tissues and organs of strawberry. Flowers (pre-fertilization); young GAPDH2. Data are mean ± SD (n = 3) and are representative of simi-
fruits (2–4  days after anthesis); big fruits (10–13  days after anthe- lar results from three independent experiments

expressed among all tissues, FvLOG5 had the highest
expression in leaves, and FvLOG1 was expressed mainly
in stolons and leaves. FvLOG2 had the highest expression
level in leaves, whereas FvLOG9 had the highest expres-
sion level in big fruits.
Expression of FvIPT and FvLOG genes in leaves
and roots under osmotic stress and high temperature
Analysis of the expression of FvIPT and FvLOG revealed
that these genes could be significantly downregulated by
exposure to osmotic stress and high temperatures (Figs. 4,
5). FvIPT5 and FvIPT6 expression was repressed in roots
and leaves after 3 and 8 h of osmotic stress and exposure to
high temperatures (Figs. 4a, b, 5a, b).
The expression levels of FvLOG2 and FvLOG9 were
near the detection limit in roots during the osmotic and
high-temperature treatments (Figs. 4d, 5d). FvLOG6 was
down-regulated in leaves and roots by osmotic stress
and high temperatures; similarly, FvLOG1 expression
decreased in leaves and roots, and FvLOG9 expression
decreased in leaves after osmotic treatment (Figs.  4c, d,
5c, d). FvLOG5 expression decreased in both roots and
leaves during the high-temperature treatment. In con-
trast, FvLOG2 was up-regulated in leaves at 8  h during
the high-temperature treatment (Fig.  5c, d). Under the
osmotic treatment, the expression of FvLOG5 decreased
at 3  h and then increased at 8  h in leaves; however, it
increased to its peak at 3 h and decreased to the untreated
control level at 8 h in roots (Fig. 4c, d).

Fig. 4  Expression profiles of
FvIPTs (a, b) and FvLOGs
(c, d) in the leaves (a, c) and
roots (b, d) of strawberry
seedlings under osmotic stress.
For osmotic stress, 3-week
old seedlings grown in 1/5
Hoagland solution supple-
mented with 25% polyethylene
glycol (PEG) 6000. Expression
data were normalized by that of
the reference gene, GAPDH2.
Data are mean ± SD (n = 3) and
are representative of similar
results from three independent
experiments

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146 Plant Growth Regul (2017) 82:139–149

Fig. 5  Expression profiles of
FvIPTs (a, b) and FvLOGs (c,
d) in the leaves (a, c) and roots
(b, d) of strawberry seedlings
under high temperature condi-
tions. For high temperature
treatment, the temperature was
elevated to 38 °C. Expression
data were normalized by that of
the reference gene, GAPDH2.
Data are mean ± SD (n = 3) and
are representative of similar
results from three independent
experiments

Expression of FvIPT and FvLOG genes in leaves
and roots treated by exogenous ABA
The expression of FvIPT and FvLOG genes was also
influenced by ABA treatment (Fig.  6). Within 0.5  h of
ABA treatment, FvIPT5 expression decreased in leaves,
but returned to the baseline level after 1 h (Fig. 6a). The
expression of FvIPT6 in leaves and roots was still down-
regulated 1  h after treatment with ABA (Fig.  6a, b).
The mRNA levels of FvLOG1, FvLOG5, and FvLOG6
genes in roots were suppressed after ABA treatment
(Fig.  6d). The expression levels of FvLOG2, FvLOG6,
and FvLOG9 also decreased in leaves after 1  h of ABA
treatment (Fig. 6c).

Fig. 6  Expression profiles of
FvIPTs (a, b) and FvLOGs
(c, d) in the leaves (a, c) and
roots (b, d) of strawberry
seedlings with ABA treatment.
The concentration of ABA is
100 μM. Expression data were
normalized by that of the refer-
ence gene, GAPDH2. Data are
mean ± SD (n = 3) and are repre-
sentative of similar results from
three independent experiments

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Plant Growth Regul (2017) 82:139–149 147
Discussion
We identified seven FvIPT genes and nine FvLOG genes
from the woodland strawberry genome. The structures,
conserved domains, and phylogenetic relationships of the
FvIPT and FvLOG genes were analyzed using bioinfor-
matics, and the patterns of tissue-specific expression and
stress-responsive expression were investigated by qRT-
PCR. These data help elucidate the function of FvIPT
genes and FvLOG genes in the regulation of growth and
stress tolerance.
Active isoprenoid CKs from seed plants are found
mainly in three forms: iP, tZ, and cZ (Sakakibara 2006;
Lomin et  al. 2015). cZ is produced primarily by tRNA-
IPT. The tRNA-IPT gene was first discovered in bacteria,
Physcomitrella patens, and some other lower organisms
(Yevdakova and Von 2007). In monocot plants, such as
maize and rice, cZ is an abundant and biologically active
cytokinin (Takagi et  al. 1985; Vyroubalova et  al. 2009).
Phylogenetic trees revealed that tRNA-IPTs are evolution-
ally conserved, and each is represented by only two genes
(AtIPT2, AtIPT9, OsIPT9, OsIPT10, FvIPT2, and FvIPT7)
from monocots (rice) to dicots (Arabidopsis and straw-
berry). However, the adenylate IPTs that are involved in
the synthesis of iP and tZ are expanded within species. It is
possible that higher plants need to regulate cytokinin bio-
synthesis more efficiently. CK receptors in Arabidopsis are
most sensitive to iP and tZ, whereas AHK3 recognizes cZ
and dihydrozeatin with relatively low sensitivity (Spichal
et al. 2004; Lomin et al. 2015). Therefore, the iP- and tZ-
type CKs are likely the major active forms in at least some
dicot plants (Lomin et al. 2015).
Various phytohormones regulate plants’ ability to coun-
teract or adapt to various abiotic and biotic stresses. Alter-
ation in CK content in plants exposed to various stresses
has been frequently reported (Tran et al. 2007; Zwack and
Rashotte 2015). Under drought stress, the content of tZ-
type CKs is significantly reduced (O’Brien and Benková
2013). As drought progresses, the bioactive CK content
in leaves gradually decreases, and is maintained for longer
periods in the upper leaves of all tested genotypes (Rivero
et al. 2007). A growing body of evidence suggests that, to
cope with water deficits, plants can activate acclimatizing
mechanisms by repressing CK biosynthesis and downregu-
lating the expression of certain components of CK signal-
ing (Li et al. 2016).
Throughout our investigation, we placed particular
emphasis on expression in response to osmotic, ABA, and
temperature stress. Previous research found that osmotic
treatment decreased the level of cytokinins in Arabi-
dopsis, in part through down-regulation of multiple IPT
genes (Nishiyama et  al. 2011). Although none of FvIPT
genes was found to contain putative drought-responsive
cis-regulatory elements, our expression analysis data
suggest that FvIPT5 and FvIPT6 gene expression was
suppressed by osmotic stress. Even though FvIPT6 and
FvLOG5 and FvLOG6 genes do not contain heat-respon-
sive cis-regulatory elements, we observed a reduction
in the expression of these genes with high-temperature
treatment. We speculate that, during osmotic and high-
temperature stresses, strawberry CK content is decreased
through down-regulation of the CK-biosynthetic FvIPT
and FvLOG genes.
The expression of CK-biosynthetic FvIPT and FvLOG
genes was down-regulated in roots by exogenous ABA
treatment (Fig. 6). Consistent with our own observation,
Vaseva et al. (2008) found that exogenous ABA treatment
decreased CK content in peas. Under stress conditions,
ABA might also participate in down-regulating CK lev-
els. In the present study, we identified a putative ABA-
responsive element in the promoter region of FvLOG5
and observed reduced FvLOG5 expression in roots after
ABA treatment. A positive drought-responsive element
was found in FvLOG6, and a heat-responsive element
was found in FvLOG9; osmotic and heat stresses reduced
the expression of FvLOG6 and FvLOG9, respectively.
Thus, the results we obtained were in contrast to those we
expected. It is clear that results of bioinformatic analy-
ses to identify cis-regulatory elements in 5′ untranslated
regions may be inconsistent with experimental data; thus,
computer-predicted putative cis-elements require experi-
mental confirmation.
Cytokinin synthesis was previously believed to be
restricted mainly to the roots, and CKs were thought to
be transported to shoots via the xylem (Beveridge et  al.
1997; Hirose et  al. 2008). However, recent studies have
demonstrated that various plant organs are capable of
synthesizing CKs (Kudo et  al. 2010). Our study showed
that FvLOG6 is expressed in numerous organs, including
roots, leaves, stems, flowers, and fruits (Fig.  3b). These
results also suggest that CKs may be produced in various
strawberry plant organs. Although we observed relatively
high expression levels of FvIPTs in roots, transcripts of
most FvLOGs were abundant in shoots, and especially
in leaves. Regardless of the effects of amplification effi-
ciency on gene expression levels, discrepancies in the
expression level of IPT and LOG may play an important
role in regulating the production of various cytokinin
species differentially in underground and aboveground
strawberry organs.
Acknowledgements  This work was supported financially by
the National Natural Science Foundation of China (31471860 and
31672124), and the Open Project of State Key Laboratory of Crop
Genetics and Germplasm Enhancement (ZW2014008).
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 Author's personal copy
148
Compliance with ethical standards  Li W, Herrera-Estrella L, Tran LS (2016) The yin-yang of cytokinin
Conflict of interest  All authors read and agreed with the final manu-
script and have no conflicts of interest.
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