The Horticulture Journal 85 (2): 128–140. 2016.
doi: 10.2503/hortj.MI-085

The Japanese Society for
JSHS

Horticultural Science

http://www.jshs.jp/

Isolation and Functional Analysis of Two Gibberellin 20-Oxidase Genes from
Satsuma Mandarin (Citrus unshiu Marc.)
Nobuhiro Kotoda1,2*, Satoshi Matsuo3, Ichiro Honda4, Kanako Yano1 and Tokurou Shimizu1
1

NARO Institute of Fruit Tree Science, Shizuoka 424-0292, Japan
Faculty of Agriculture, Saga University, Saga 840-8502, Japan
3
NARO Institute of Vegetable and Tea Science, Tsu 514-2392, Japan
4
Department of Biotechnology, Maebashi Institute of Technology, Maebashi 371-0816, Japan
2

Satsuma mandarin (Citrus unshiu Marc.) has two GA 20-oxidase genes, CuGA20ox1 and CuGA20ox2, and the
genomic sequence for CuGA20ox1 is shorter than that for CuGA20ox2, although the coding region of cDNA for
CuGA20ox1 is slightly longer than that for CuGA20ox2. Southern blot analysis revealed that 12 Citrus
cultivars examined and trifoliate orange [Poncirus trifoliata (L.) Raf.] have at least two types of GA 20-oxidase
genes, such as CuGA20ox1 and CuGA20ox2 genes. CuGA20ox1 and CuGA20ox2 were differentially expressedin various tissues. CuGA20ox1 was expressed in almost all of the tissues investigated with relatively higher
expression in vegetative than in reproductive tissues, whereas CuGA20ox2 was specifically expressed in flower
buds just before anthesis. These distinct expression patterns of CuGA20ox1 and CuGA20ox2 imply thatfunction of these two genes diverged in the process of evolution. The specific and relatively higher expressionof CuGA20ox2 in flower buds would explain why GA-like activity was higher in Satsuma mandarin’s ovaries
at anthesis. Transgenic Arabidopsis [Arabidopsis thaliana (L.) Heynh] plants ectopically expressing CuGA20ox1
or CuGA20ox2 were examined to elucidate the function of these two Satsuma mandarin genes. Phenotypic
analysis revealed that both CuGA20ox1 and CuGA20ox2 caused elongated inflorescence but did not affect thetiming of flowering in transgenic Arabidopsis as compared with wild-type controls. Ectopic expression of
CuGA20ox1 and CuGA20ox2 significantly affected the levels of GA24 and GA34 on the non-13-hydroxylation
pathway; GA24 decreased and GA34 increased. This observation indicates that both CuGA20ox1 andCuGA20ox2 accelerated the conversion of GA24, a substrate of a GA 20-oxidase, to GA9, a precursor of anactive form of GA4. Likewise, on the early-13-hydroxylation pathway, ectopic expression of CuGA20ox1
significantly decreased GA 19 and increased GA 29 and GA8, inactive metabolites of 2-hydroxylation of GA20 and
GA1, respectively, suggesting the activation of this biosynthetic pathway. CuGA20ox2 also had a tendency toactivate the early-13-hydroxylation pathway although it increased only GA20 with a statistically significant
difference. Taken together, we concluded that CuGA20ox1 and/or CuGA20ox2 activated both the early-13- andnon-13-hydroxylation pathways for increasing active GAs, resulting in elongated inflorescences in transgenic
Arabidopsis.
Key Words: CuGA20ox1, CuGA20ox2, LC-MS/MS, plant growth substances, seedless fruit.

Introduction
Gibberellins (GAs) are plant growth substances, origReceived; June 4, 2015. Accepted; September 23, 2015.
First Published Online in J-STAGE on November 28, 2015.
This study was supported by a Grant-in-Aid for scientific researchfrom the Ministry of Education, Culture, Sports, Science andTechnology (No. 24580058) and a Research Project for Molecular
Evaluation of Parthenocarpy on Horticultural Crops from NARO.
* Corresponding author (E-mail: koto@cc.saga-u.ac.jp; nobu_fruits@yahoo.co.jp).

inally identified in a rice bakanae fungus [Gibberella
fujikuroi (Sawada) Wollenw.] as a toxin that caused rice
to elongate, resulting in lodging and low yields(Kurosawa, 1926; Yabuta and Sumiki, 1938). In the late
1950s, the chemical structure of GA3 was determined(Cross, 1960; Cross et al., 1959; Takahashi et al., 1961)
and total synthesis of some GAs was achieved in the
1960s to 1970s (Corey et al., 1978; Mori et al., 1969),
consequently leading to their use in biochemical studies
and agricultural practices. Kawarada and Sumiki (1959)
reported that Satsuma mandarin (Citrus unshiu Marc.)© 2016 The Japanese Society for Horticultural Science (JSHS), All rights reserved.

Hort. J. 85 (2): 128–140. 2016.

contained GA1 in its water sprouts (vegetative shootsthat produce neither flowers nor fruit), and first demonstrated the existence of a GA in woody plants.
Extensive studies for many years revealed that GAs
are involved in a wide variety of physiological events,
such as cell elongation, cell division, dormancy, flowering, and fruit development in higher plants. Many researchers have also conducted biochemical studies toclarify the biosynthetic pathway of GAs in plants (forreviews: Hedden and Kamiya, 1997; Hedden andPhillips, 2000; Yamaguchi, 2008). These studies revealed that there are two pathways for synthesizing active GAs; the pathway where GAs, such as GA1 andGA3, are synthesized sequentially through GA53, GA44,
GA19, and GA20 after the synthesis of GA12 throughsome steps from isopentenyl diphosphate and dimethylallyl diphosphate, and another pathway where GAs,such as GA4, are synthesized through GA15, GA24, andGA9 after the synthesis of GA12 (Fig. 1). The former
and the latter pathways are known as the early-13hydroxylation and the non-13-hydroxylation pathways,
respectively, because GAs in the former pathway arehydroxylated at position C13 in the early step from
GA12 to GA53, whereas those in the latter are not. As in
rice, citrus, such as Satsuma mandarin, is considered touse the early-13-hydroxylation pathway because citrus
accumulates 13-OH GAs, such as GA1, more abundantly than it accumulates 13-H GAs, such as GA4 (Gotoet al., 1989; Poling and Maier, 1988; Talon et al.,1990a, b; Turnbull, 1989). Recently, the genes encoding
the enzyme to catalyze the step from GA12 to GA53,
which have been unknown for many years, were identified as CYP714B1 and CYP714B2 in rice (Oryza sativa

129L.) (Magome et al., 2013). In the rice double mutant,
cyp714b1 cyp714b2, the concentration of 13-H GAs
that include GA4 increased, on the other hand, transgenic rice overexpressing CYP714B1 or CYP714B2
increased the level of 13-OH GAs that include GA1,
which show semi-dwarfism. This result suggests thatthese GA 13-oxidases may be involved in the switchingof the two biosynthetic pathways that diverge after
GA12.
Also in woody plants like fruit trees, much effort has
been devoted to understanding the role of GAs. It has
been demonstrated, for example, that in apple (Malus
domestica Borkh.) and citrus GAs function negatively
to flower induction (Luckwill and Silva, 1979;
Monselise, 1979; Tromp, 1982). On the other hand, it isreported that GAs play a positive role in the floweringof Japanese cedar (Cryptomeria japonica), although themechanism underlying the phenomenon remains to beclarified (Nagao et al., 1989). The method for producing seedless grapes by applying GA3 was established as
early as the structure of GA3 was determined in the late
1950s. Kishi and Tasaki (1960) found that berries matured early and were seedless when their flower buds
were treated with adequate concentrations of GA3 about
three weeks before full bloom in ‘Delaware’ grape,
which was originally a seeded cultivar. These findings
were applied commercially to the culture of seedlessgrapes in Japan for the first time in the world.
Production of seedless fruit is very important forbreeders and growers to meet consumer demands incitriculture as well as in viticulture. Satsuma mandarin
is considered absolutely seedless because it is female
and male sterile in addition to parthenocarpic and very

Fig. 1. Biosynthetic pathway of gibberellin in higher plants. IPP, isopentenyl diphosphate, GA20-ox, GA 20-oxidase; GA3-ox, GA 3-oxidase;
GA2-ox, GA 2-oxidase. Boxes in gray represent active GAs and dotted boxes represent inactive GAs.

130

N. Kotoda, S. Matsuo, I. Honda, K. Yano and T. Shimizu

productive without pollinators (Iwamasa, 1966; Vardi
et al., 2008). Talon et al. (1990b) reported that in
Satsuma mandarin the development of ovaries precededthat in Clementine (Citrus clementina hort. ex Tanaka),
which shows self-incompatibility and low parthenocarpy. In addition, active GAs and its precursors, suchas GA19, GA20, and GA1, were more highly accumulated in the ovaries at around anthesis in Satsuma mandarin than in Clementine (Talon et al., 1990b, 1992).
These results imply a relationship between the degreeof early fruit (ovary) development and parthenocarpy
and the endogenous GAs there. In recent years, genes
related to GA biosynthesis have been isolated and analyzed to clarify their functions in citrus (Fagoaga et al.,2007; Huerta et al., 2009; Vidal et al., 2001, 2003).
Fagoaga et al. (2007) demonstrated that transgenic
Carrizo citrange [a hybrid of trifoliate orange and sweetorange (Citrus sinensis Osbeck)] that overexpressed
CcGA20ox1 (a Carrizo citrange GA 20-oxidase gene)increased the GA1 level and the height of the plant. In
addition, Huerta et al. (2009) identified and characterized the second citrus GA 20-oxidase gene,
CcGA20ox2, in Carrizo citrange. In this study, we report the characterization of two GA 20-oxidase genes
from Satsuma mandarin, which is cultivated in the largest area in the citriculture of Japan, as a first step inclarifying the roles of these genes and the distributionof GAs in the life cycle of citrus, such as Satsuma mandarin. Satsuma mandarin originated in Japan, and is aunique and important scion cultivar providing us withseedless and easy-peeling fruit with health functional
ingredients for over 150 years. Molecular characterization of GA biosynthetic genes including Citus unshiu
GA-20 oxidase genes would be helpful to studying themechanisms of parthenocarpy and peel puffins in the
future.

Materials and Methods
Plant materials
Tissue samples of a Satsuma mandarin (Citrus unshiu
Marc.) cultivar (cv.) ‘Silverhill’ in the adult phase (age:43 yr) were collected from the experimental field at the
NARO Institute of Fruit Tree Science in Okitsu,
Shizuoka, Japan. Two-year-old nucellar seedlings of‘Silverhill’, which were distinguished from hybrids by
simple sequence repeat (SSR) genotyping analysis,
were used for expression analysis in the juvenile phase.
Satsuma mandarins (cvs. ‘Silverhill’ and ‘Miyagawawase’), ‘Kiyomi’ tangor (C. unshiu × C. sinensis
Osbeck), Hyuganatsu (C. tamurana hort. ex Tanaka),
Clementine, ‘Clementine Vita’ (Citrus clementina hort.
ex Tanaka cv. ‘Clementine Vita’), sweet orange(C. sinensis Osbeck cv. ‘Trovita’), pummelo (C. grandis
Osbeck cvs. ‘Benimadoka’ and ‘Banpeiyu’), Sudachi(C. sudachi hort. ex Shirai), Yuzu (C. junos Sieb. ex
Tanaka), Kunenbo (C. nobilis Lour.), and trifoliate orange [Poncirus trifoliata (L.) Raf.] were used forSouthern blot analysis. Wild-type plants of ecotype
Columbia (Col) were used for Arabidopsis [Arabidopsis
thaliana (L.) Heynh] transformation.
Isolation of CuGA20ox1 and CuGA20ox2 genes from
Satsuma mandarin
Leaves and ovaries of Satsuma mandarin ‘Silverhill’
were used to isolate the cDNA of GA 20-oxidase genes
CuGA20ox1 and CuGA20ox2, respectively. cDNA fromthe leaves and ovaries was amplified by polymerase
chain
reaction
(PCR)
with
the
primers
CcGA20ox1(−24 > −3) and CcGA20ox1(1239 < 1258)
for CuGA20ox1 and CcGA20ox2(−29 > −4) andCcGA20ox2(1124 < 1147) for CuGA20ox2, designed
based on the sequences of CcGA20ox1 (AJ250187;
Vidal et al., 2001) and CcGA20ox2 (EU834066; Huerta
et al., 2009), respectively, using KOD-plus (Toyobo,
Osaka, Japan). Each amplified product was cloned intoa pT7Blue T-vector (Merck Millipore, Darmstadt,
Germany) or a pGEM-T Easy vector (Promega,
Madison, WI, USA) after an adenine nucleotide addition to the products. To obtain genomic DNA of
CuGA20ox1 and CuGA20ox2, the same primer sets as
those for cDNA isolation were used with the total DNA
of Satsuma mandarin ‘Silverhill’ as a template. All
PCR products for the isolation of cDNA and genomic
DNA of CuGA20ox1 and CuGA20ox2 were subcloned
and sequenced (ABI 3130xl DNA sequencer; Life
Technologies, Carlsbad, CA, USA). The primer sets
used in gene cloning are listed in Table 1.
Construction of a phylogenetic tree
Amino acid sequences were analyzed using theClustalX multiple sequence alignment programver. 1.83 (Jeanmougin et al., 1998) and BioEdit
ver. 7.7.0 (Hall, http://www.mbio.ncsu.edu/BioEdit/
bioedit.html). The tree was constructed by theneighbor-joining (N-J) method for the deduced aminoacid sequence of the GA 20-oxidase genes from apple[MdGA20ox(AB037114),
MdGA20ox(MDP0000136940)],
Arabidopsis[AtGA20ox1(AT4G25420), AtGA20ox2 (AT5G51810), AtGA20ox3(AT5G07200), AtGA20ox4 (AT1G60980), AtGA20ox5(AT1G44090A)], Carrizo citrange [Citrus sinensis ×
Poncirus
trifoliata,
CcGA20ox1(AJ250187),
CcGA20ox2 (EU834068)], Clementine [CclGA20ox1(Ciclev10005157m),
CclGA20ox2(Ciclev10020694m)], common bean [Phaseolus
vulgaris L., PvGA20ox (U70530, U70531, U70532) ],European beech [Fagus sylvatica L., FsGA20ox1(AJ420192)], grape [Vitis vinifera L., VvGA20ox(DQ508817)], lettuce [Lactuca sativa, LsGA20ox1(AB012203), LsGA20ox2 (AB012204), LsGA20ox3(AB031202)], morning glory [Ipomoea nil (L.) Roth,InGA20ox1 (AB099488), InGA20ox2 (AB099489)],pea [Pisum sativum L., PsGA20ox (U70471),
PsGA20ox (U58830)], Physcomitrella patens (Hedw.)

Table 1. Primer sets used in gene cloning, probe labelling, quantitative real-time RT-PCR analysis, and vector construction.
Hort. J. 85 (2): 128–140. 2016.

131

Table 1. Primer sets used in gene cloning, probe labelling, quantitative real-time RT-PCR analysis, and vector construction.
Primers

Oligonucleotide (5′ → 3′)

Gene cloning
CcGA20ox1(-24 > -3)
CcGA20ox1(1239 < 1258)
CcGA20ox2(-29 > -4)
CcGA20ox2(1124 < 1147)

CTC AAC TCA CAA CTC TTT AAT G
TTG CTA GCC CTT TGT CTT CCACC CAT CTA TCT ATT ACA ATC TAT CT
CAT CAT CTT AAA TTT CCT ATT CAC

Probe labelling
CcGA20ox1(-24 > -3)
CcGA20ox1(1239 < 1258)
CuGA20ox2(1 > 26)
CuGA20ox2(1096 < 1119)
Real-time RT-PCR
CuGA20ox1(522 > 543)
CuGA20ox1(627 < 646)
CuGA20ox2(481 > 505)
CuGA20ox2(578 < 600)

CTC AAC TCA CAA CTC TTT AAT G
TTG CTA GCC CTT TGT CTT CC
ATG TCA ATG GAT TGT TAC AAT TCA AG
TTA AAT ATT GAC ATG CTG TTG AAG

CuActin(-86 > -66)
CuActin(-1 < 19)
AtTUB4-F

GCT TAA CAC AAT GGG AGA TGA A
CCC TGT CTA CAC CAA GAC TT
GTC GAA GAT TAT TTC TTC AAC GTA A
CAT TCC TAA CAG CTC CAT GAT CC
GAG CGA TAG AGA GAA TCG ACATAT CCT CAG CAT CGG CCA TT
TTC ATA TCC AAG GCG GTC AAT GTG G

AtTUB4-R

CGA GCT TGA GGG TAC GGA AAC AG

Vector construction
CuGA20ox1(1 < 20)XbaI
CuGA20ox1(1131 < 1152)SacI
CuGA20ox2(1 > 25)XbaI
CuGA20ox2(1095 < 1119)SacI

AAt cta gaA TGG CAA TAG ACT GCA TAA A
AAg agc tcT CAT TTC AGC TGT TTT TTC TGT
AAt cta gaA TGT CAA TGG ATT GTT ACA ATT CAA
AAg agc tcT TAA ATA TTG ACA TGC TGT TGA AGC

The thermal cycle programs for real-time RT-PCR were as follows: 95°C for 10 min, followed by 40 cycles of at 95°C for 15 s, 60°C for 60 s for
CuGA20ox1 [CuGA20ox1(522 > 543) and CuGA20ox1(627 < 646)], CuGA20ox2 [CuGA20ox2(481 > 505) and CuGA20ox2(578 < 600)], CuActin[CuActin(-86 > -66) and CuActin(-1 < 19)] and AtTUB4 (AtTUB4-F and AtTUB4-R). Characters in lowercase indicate the site for restriction
enzymes used for vector construction.

Bruch & Schimp [PpGA20ox (EU262757)], poplar[Populus nigra L., PnGA20ox1 (AB089675); P. AlbaL.,
PaGA20ox1a(AB090238);
P. tremula ×
P. tremuloides Michx., PtPtGA20ox1 (AJ001326)],
potato [Solanum tuberosum L., StGA20ox1 (AJ291453),
StGA20ox2 (AJ291454)], Selaginella moellendorffii[SmGA20ox (EU262754)], rice [Oryza sativa L.,
OsGA20ox1 (U50333), OsGA20ox2 (AB469066)],
Satsumamandarin[CuGA20ox1(LC056054),
CuGA20ox2 (LC056055)], spinach [Spinacia oleracea
L., SoGA20ox (U33330)], sweet orange [Citrus
sinensis,
CsGA20ox1(orange1.1g016776m),
CsGA20ox2 (orange1.1g041132m)], tobacco [Nicotiana
tabacum L., NtGA20ox (AB012856); N. Sylvestris
Speg. & Comes, NsGA20ox2 (AF494088)], and tomato[Solanum lycopersicum L., LeGA20ox1 (AF049898),
LeGA20ox2 (AF049899), LeGA20ox3 (AF049900),
SlGA20ox4 (EU675630)]. The phylogenetic tree wasdisplayed using the N-J plot unrooted (Perrière andGouy, 1996) with bootstrap values for 1000 resamplings in each branch.
DNA extraction and hybridization analysis
Genomic DNA was isolated using a cetyltrimethylammonium bromide (CTAB)-based method as de-scribed in Kotoda et al. (2002). For Southern blot
analysis, the genomic DNA (5 μg) was digested with
XbaI, electrophoresed on a 0.8% agarose gel, and thenblotted onto Hybond-N+ nylon membranes (GEHealthcare Bio-Sciences Corp., Piscataway, NJ, USA).
The blotted membrane was hybridized with digoxigenin(DIG; Roche Diagnostics, Mannheim, Germany)labeled CuGA20ox1 or CuGA20ox2 cDNA, which was
amplified by PCR with a pair of primers used for genecloning for CuGA20ox1 and with a pair of primers
CuGA20ox2(1 > 26) and CuGA20ox2(1096 < 1119) for
CuGA20ox2 and a DIG mixture (Roche Diagnostics).
Hybridization was performed in DIG Easy Hyb (Roche
Diagnostics) at 42°C for 16 h, followed by two rinses in
2x SSC containing 0.1% (w/v) SDS at room temperature for 2 min and two washes in 0.5x SSC containing
0.1% (w/v) SDS at 68°C for 20 min as described by
Kotoda et al. (2006). Detection was performed according to the manufacturer’s protocol (Roche Diagnostics).
Chemiluminescent signals were visualized using theNightOWL image analyzer (Berthold Technologies,
Bad Wildbad, Germany). The primer sets used forlabelling are listed in Table 1.

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N. Kotoda, S. Matsuo, I. Honda, K. Yano and T. Shimizu

Expression analysis by quantitative real-time RT-PCR
Using a phenol-SDS based method as described by
Ikoma et al. (1996), total RNA was extracted for analysis from seeds, roots (whole roots including lateralones), stems, new and old leaves, and nodes of twoyear-old juvenile nucellar seedlings as well as from new
and old leaves, shoot apexes in August, flower buds
(just before anthesis), young fruit in June, juice sacs (inSeptember, November, and December), and peels (inSeptember and December) of adult ‘Silverhill’ Satsuma
mandarin trees. For transgene expression analysis,
whole plants of transformants in second generation (T2)and controls were collected two months after incubation, and total RNA was extracted from two individuals
per each line by using a CTAB based methods. The
samples of total RNA were then purified using an
RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany).
First-strand cDNA was synthesized from 1 μg of total
RNA in 20 μL of a reaction mixture using a QuantiTect
Reverse Transcription Kit (Qiagen). Subsequent PCR
reactions were performed with 1 μL of the first-strand
cDNA as a template in a total volume of 12.5 μL using
the ABI PRISM® 7000 (Applied Biosystems, Foster
City, CA, USA). Transcripts of CuGA20ox1,
CuGA20ox2, an actin gene derived from Satsuma mandarin (CuActin; for reference, Phytozome Transcript
Name Ciclev10025866m), and an Arabidopsis β tubulin
gene (AtTUB4) were identified with the specific primers. The primer sets used in the quantitative real-time
RT-PCR (qRT-PCR) analysis and the PCR conditions
are described in Table 1. A citrus CuActin gene and an
Arabidopsis AtTUB gene were used as an internal control for analyzing Satsuma mandarin and Arabidopsis,
respectively. qRT-PCR was performed three times, andtranscript levels were normalized against each internal
control.
Construction of the transformation vector
To construct a vector for the constitutive expressionof CuGA20ox1 and CuGA20ox2, the coding region ofeach gene was amplified by PCR with pairs of primers
CuGA20ox1(1 < 20)XbaI and CuGA20ox1(1131 <1152)SacI for CuGA20ox1, and with a pair of primers
CuGA20ox2(1 > 25)XbaI and CuGA20ox1(1095 <
1119)SacI for CuGA20ox2. An amplified PCR productwas subsequently digested with XbaI and SacI, and thencloned into the XbaI/SacI sites of the modified
pSMAK193E (35SΩ/pSMAK193E; described in
Kotoda et al., 2010), a kanamycin-resistant binary vector, to be placed between the 35SΩ and 3' regions of the
Arabidopsis rbcS-2B gene (TrbcS).
Arabidopsis transformation
Agrobacterium tumefaciens strain EHA101 harboringa binary vector with 35SΩ::CuGA20ox1 or
35SΩ::CuGA20ox2 was used to transform Arabidopsis
thaliana (Col) plants using the floral-dip method(Clough and Bent, 1998). After two to five adult leaves
had developed, kanamycin-resistant transformants weretransplanted from the plate to moistened potting soil
composed of vermiculite and grown in a growth chamber (Biotron; Nippon Medical and Chemical
Instruments Co., Ltd., Tokyo, Japan) set at 22°C under
LD conditions (16-h photoperiod; cool white fluorescent light, 50 μmol·m−2·s−1). Morphological and expression analyses and quantification of gibberellins (GAs)were performed on the second generation (T2). For morphological analysis, numbers of rosette and cauline
leaves were counted on the day floral organs became
visible. Numbers of inflorescences merging from thebase were counted and length of 1st inflorescences(infl.) was measured one month after incubation. Whole
plants of transformants (T2) and controls were collected
two months after incubation for qRT-PCR analysis described above and quantification of gibberellins (GAs)described below.
Quantitative analysis of gibberellins
Two month after the start of incubation, whole transgenic and wild-type Arabidopsis plants were used forthe quantification of 13 gibberellins on both theearly-13-hydroxylation pathway (GA53, GA44, GA19,
GA20, GA1, GA8, and GA29) and the non-13hydroxylation pathway (GA15, GA24, GA9, GA4, GA34,
and GA51). Five plants (about 0.4–1.2 g in total) per
each transgenic line or control were collected and frozen in liquid nitrogen. These plants were then groundwith a pestle in liquid nitrogen and homogenated in
80% methanol, followed by the adition of [17, 17-2H2]
GAs (5 ng each) as internal standards. A total of ten
samples (one sample per each independent line of
35S::CuGA20ox1 or 35S::CuGA20ox2) were preparedin this experiment (n = 5, five biological replicates).
After incubation for 12 h at 4°C, each homogenate wascentrifuged and supernatant was concentrated by evaporating the methanol. The concentrate was partitioned
against an equal volume of n-hexane, and the aqueous
layer was obtained, the pH of which was adjusted to
3.0. The aqueous layer was partitioned with ethyl acetate and the organic layer was obtained (three times).
Then, the organic solvent was extracted three times
with potassium phosphate buffer (pH = 8.3). The pH of
the aqueous extracts was adjusted to/below 3.0 again,
and the extracts were partitioned with ethyl acetate;then, an organic layer was obtained, followed by drying
with sodium sulfate. The dried organic solvent was concentrated to evaporate the ethyl acetate after filtration
through a Bond Elut® Reservoir (Varian, Lake Forest,
CA, USA). The sample dissolved in methanol was
loaded onto a reverse phase cartridge (Oasis® HLB,
60 mg; Waters, Milford, MA, USA) and eluted with
80% methanol. The elute was dried and then the sample
in methanol was loaded onto an ion-change column(Bond Elut® DEA; Varian). GAs were eluted three133

Hort. J. 85 (2): 128–140. 2016.

times with 2 mL of methanol containing 2% acetic acid,and the elute was dried, dissolved in chloroform:ethyl
acetate = 1:1 (v/v) containing 1% acetic acid, and thenloaded onto a Sep-Pak® silica cartridge (500 mg;
Waters). GAs were eluted with chloroform:ethyl acetate = 1:1 (v/v) containing 1% acetic acid and the elute
was concentrated to dryness, dissolved in 50 μL of 20%
methanol containing 0.05% acetic acid, filtered througha PTFE membrane, and then subjected to LC-MS/MS
analysis. The LC-MS/MS system consisted of a
Shimadzu Prominence 20A Series HPLC (Shimadzu,
Kyoto, Japan) equipped with a 3200 QTRAP® LCMS/MS System (AB Sciex, Framingham, MA, USA),using an electrospray ionization (ESI) interface. The
purified samples were injected onto a Shim-pack XRODS column (2.2 μm, 75 × 2.0 mm; Shimadzu) at
45°C, and eluted at a flow rate of 0.2 mL·min−1. Mobile
phase A, consisting of water/methanol/acetic acid(80:19.95:0.05, v/v/v), and mobile phase B (methanol)
were used for chromatographic separation. The initialcondition was 100% A, which was maintained for
2 min, changing linearly to 40% A and 60% B after
5 min, and changing to 100% B after 10 min, and finally maintained at 100% B for 6 min. The column was
equilibrated
with the starting composition of the mobile
Figure2 Kotoda
phase for 12 min before each analytical run. Quantifica-A

B

CuGA20ox1

557

CuGA20ox2

530

126

234

tion of the GAs was described previously (Fukuda
et al., 2009).
Statistical analysis
Data were analyzed using a one-way ANOVA andthe differences were contrasted using Tukey’s multiple
comparison. All statistical analyses were perfomed at asignificance level of P-value < 0.05 using R-3.2.0 (RCore Team, 2015).

Results
Isolation of CuGA20ox1 and CuGA20ox2 from Satsuma
mandarin
To investigate the function of GA 20-oxidase genes
in Satsuma mandarin, we isolated both cDNA and genomic DNA of these genes from Satsuma mandarin‘Silverhill’ by polymerase chain reaction (PCR) withprimer sets based on the sequences of CcGA20ox1(AJ250187) and CcGA20ox2 (EU834066) (Huerta
et al., 2009; Vidal et al., 2001). Coding sequences ofCitrus unshiu GA20ox1 (CuGA20ox1; Accession
No. LC056054) and Citrus unshiu GA20ox2(CuGA20ox2; Accession No. LC056055) cDNA were1152 and 1119 bp long including a stop codon and encoded 383 and 372 amino acids, respectively (Fig. 2A).
Identities at the nucleotide level between CuGA20ox1

322

322

131

108

272

267

100 bp

10
20
30
40
50
60
70
80
90
100
110
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. . ..| ....| .. .. |.. .. |.. .. | . ... |. .. . |. .. .|. .. .|. . . . | .. . . | .. . . | . . .. | . . . . |. . . . | . . . . |. . . . |. . . . | . . . . | . . . . | . .. . | . . . .| . . . . | . . . .| . . . . | . . .. |CuGA20ox1 MAIDCIKNIPTMLHQPKEDYKDEQKPLVFDASVLKHQTQIPKQFIWPDDEKPCVNAPELQVPLIDLGGFLSDDPVAAKEASRLVGEACRKHGFFLVVNHGVDSSLIADAHRYMDHFFELPLNEKQRARRKCclGA20ox1 MAIDCIKNIPTMLHQPKEDYKDEQKPLVFDASVLKHQTQIPKQFIWPDDEKPCVNAPELQVPLIDLGGFLSDDPVAAKEASRLVGEACRKHGFFLVVNHGVDSSLIADAHRYMDHFFELPLNEKQRARRKCcGA20ox1 MAIDCIKNIPTMLHQPKEEYKDEQKPLVFDASVLKHQTQIPKQFIWPDDEKPCVNAPELQVPLIDLGGFLSDDPVAAKEASRLVGEACRKHGFFLVVNHGVDSSLIADAHRYMDHFFELPLNEKQRARRK
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. . ..| ....| .. .. |.. .. |.. .. | . ... | . .. . |. .. .|. .. . |. . . . | . .. . | .. . .| . . ..| . . . . |. . . . | . . . . |. . . . | . . . . | . . . . | . . . . | . .. .| . . . . | . . . . | . . . . | . . . .| .. . .|CuGA20ox1 LGEHCGYASSFTGRFSSKLPWKETLSFRYSAEKSLSNNIVEDYLLNTMGDEFKQFGRVYQDYCESMSRLSLGIMELLAISLGVDRAHFKEFFEENDSIMRLNYYPPCQKPELTLGTGPHCDPTSLTILHQ
CclGA20ox1 LGEHCGYASSFTGRFSSKLPWKETLSFRYSAEKSLSNNIVEDYLLNTMGDEFKQFGRVYQDYCESMSRLSLGIMELLAISLGVDRAHFKEFFEENDSIMRLNYYPPCQKPELTLGTGPHCDPTSLTILHQ
CcGA20ox1 LGEHCGYASSFTGRFSSKLPWKETLSFRYSAEKSLSNNIVEDYLLNTMGDEFKQFGRVYQDYCESMSRLSLGIMELLAISLGVGRAHFKEFFEENDSIMRLNYYPPCQKPELTLGTGPHCDPTSLTILHQ
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. . .. | .... | .. .. | .. .. | .. .. | . ... | . .. . | . .. . | . .. . | . . . . | . .. . | .. . . | . . .. | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .. . | . . . . | . . . . | . . . . | . . .

CuGA20ox1 DQVGGLQVFVENEWRSISPNFEAFVVNIGDTFMALSNGRYKSCLHRAVVNSQTTRKSLAFFLCPKNDKVVSPPSELVDTYSSPRIYPDFTWPMLLEFTQKHYRADMKTLEVFTNWLQQKKQLKCclGA20ox1 DQVGGLQVFVENEWRSISPNFEAFVVNIGDTFMALSNGRYKSCLHRAVVNSQTTRKSLAFFLCPKNDKVVSPPSELVDTYSSPRIYPDFTWPMLLEFTQKHYRADMKTLEVFTNWLQQKKQLKCcGA20ox1 DQVGGLQVFVDNEWRSISPNFEAFVVNIGDTFMALSNGRYKSCLHRAVVNSQTTRKSLAFFLCPKNDKVVSPPSELVDTYSSPRIYPDFTWPMLLEFTQKHYRADMKTLEAFTNWLQQKKQLK

10
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. .. .|. .. . | ... . | .. .. | .. .. |.. .. |. ... | . .. . | . .. . | . .. . | . . ..| . . ..| . . . . |. . . . |. . . . |. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |. . . .|. . . . |. . . .| .. . . |CuGA20ox2 MSMDCYNSSILPTQEQLLVFDASVLQFQSTIPSQFIWPDGEKPSLELPELVIPPIDLCSFLSGDPFEVDKICQLVDEACKKHGFFLVVNHGVDSMLIKKAHKYMDSFFATQLSKKQKAQRKIGDHCGYSS
CclGA20ox2 MSMDCYNSSILPTQEQLLVFDASVLQFQSTIPSQFIWPDGEKPSLELPELVIPPIDLCSFLSGDPFEVDKICQLVDEACKKHGFFLVVNHGVDSMLIKKAHKYMDSFFATQLSKKQKAQRKIGDHCGYSSCcGA20ox2 MSMDCYNSSILPTQEQLLVFDASVLQFQSTIPSQFIWPDGEKPSLELPELVIPPIDLRSFLSGDPFEVDKICQLVDEACKKHGFFLVVNHGVDSMLIKKAHKYMDSFFATQLSKKQKAQRKIGDHCGYSS
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. .. .|. .. . | ... . | .. .. | .. .. | .. .. |. ... | . .. . | . .. . | .. . .| .. . .| .. . .| . . . . | . . . . | . . . . |. . . . |. . . .| . . . . | . . . . | . . . . |. . . . |. . . . | . . . .|. . . . |. . . .| .. . . |CuGA20ox2 SFTGRFSNKLPWKETLTFRYRANDENSSTVVEDYFFNVMGQDFRQFGKVYQEYCEAMSSLSLGIMELLGMSLGVGRAHFREFFQGHDSIMRLNYYPPCQKPNLTLGTGPHCDPTSLTILHQDQVGGLQVY
CclGA20ox2 SFTGRFSNKLPWKETLTFRYRANDENSSTVVEDYFFNVMGQDFRQFGKVYQEYCEAMSSLSLGIMELLGMSLGVGRAHFREFFQGHDSIMRLNYYPPCQKPNLTLGTGPHCDPTSLTILHQDQVGGLQVY
CcGA20ox2 SFTGRFSNKLPWKETLTFRYRANDENSSTVVEDYFFNVMGQDFRQFGKVYQEYCEAMSSLSLGIMELLGMSLGVGRAHFREFFQGHDSIIRLNYYPPCQKPNLTLGTGPHCDPTSLTILHQDQVGGLQVY
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. .. . |. .. . | ... . | .. .. | .. .. | .. .. |. ... | . .. . | . .. . | . .. . | .. . . | .. . . | . . . . |. . . . |. . . . | . . . .|. . . .| . . . .| . . . . | . . . . |. . . . |. . . . | . .

CuGA20ox2 VDEQWQSVSPNPDAFVVNIGDTFMALSNGIYKSCLHRAAVNNTTVRKSLAFFLCPKMDKVVTPPNNLVNANNPRIYPDFTWHNLLHFTQNYFRADVKTLNAFSAWLQQHVNICclGA20ox2 VDEQWQSVSPNPDAFVVNIGDTFMALSNGIYKSCLHRAAVNNTTVRKSLAFFLCPKMDKVVTPPNNLVNANNPRIYPDFTWHNLLHFTQNYFRADVKTLNAFSAWLQQHVNICcGA20ox2 VDEQWQSVSPNPDAFVVNIGDTFMALSNGIYKSCLHRAAVNNTTVRKSLAFFLCPKMDKVVTPPNNLVNANNPRIYPDFTWHNLLHFTQNYFRADVKTLNAFSAWLQQHVNI

Fig. 2. Schematic representation of the genomic organization (A) and comparison of the deduced amino acid sequence (B) of CuGA20ox1 andCuGA20ox2. Black boxes represent exons and lines represent introns. Numbers represent the lengths (bp) of exons (in the boxes) and introns(below the lines). The scale bar on the map represents approximately 100 bp.

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and CcGA20ox1 and between CuGA20ox2 andCcGA20ox2 were 99.5% and 99.4%, respectively, andthose at the amino acid level were 99.0% and 99.2%,
respectively (Fig. 2B). In addition, at the amino acidlevel, CuGA20ox1 and CuGA20ox2 were identical to
CclGA20ox1 (Ciclev10005157m) and CclGA20ox2(Ciclev10020694m) from Clementine, respectively. For
genomic clones of CuGA20ox1 and CuGA20ox2, sequences including the coding region of each gene andthe exon-intron structure were identified (Fig. 2A).
Southern blots of the genomic DNA of 12 Citrus species/cultivars and trifoliate orange (Poncirus trifoliata)
digested with XbaI hybridized with DIG-labeled cDNAof CuGA20ox1 and CuGA20ox2 showed a single bandof about 10 kb for CuGA20ox1 (Fig. 3A) and polymorphic bands for CuGA20ox2 (Fig. 3B). To examine theevolutionary relationships among GA 20-oxidase genes
including CuGA20ox1 and CuGA20ox2, we performed
phylogenetic analysis of the amino acid sequences corresponding to these genes from a variety of Citrus species and Poncirus sp. (Fig. 4A). Neighbor-joining (N-J)
distance analysis generated an unrooted tree with four
clades as previously shown by Huerta et al. (2009).
CuGA20ox1 and CuGA20ox2 were classified into thedifferent two clades as GA 20-oxidases from apple,
common bean (Phaseolus vulgaris) and pea (Pisum
sativum). Among Citrus species, putative GA 20-

oxidases from Satsuma mandarin were phylogeneticallyvery similar or identical to those from sweet orange andClementine as expected (Fig. 4B).
Organ-specific expression patterns of CuGA20ox1 andCuGA20ox2
To identify the expression patterns of CuGA20ox1
and CuGA20ox2, quantitative real-time reverse
transcription-PCR (qRT-PCR) was performed on various tissues in Satsuma mandarin in juvenile and adultphases using primers specific to each gene (Table 1).
The transcript of CuGA20ox1 accumulated mainly innew (NL) and old (OL) leaves in both the juvenile andadult phases. It was also detected clearly in seeds androots, stems, and nodes of seedlings in the juvenile
phase and shoot apexes (SA), juice sacs (JS) in November and December, and peels (PL) in December, but
only faintly in flower buds (FB), young fruit (YF), andjuice sacs and peels in September (Fig. 5A). In contrast,
the transcript of CuGA20ox2 accumulated specificallyin flower buds with very low expression in peels(Fig. 5B).
Characteristics of transgenic Arabidopsis ectopically
expressing CuGA20ox1 or CuGA20ox2
To elucidate the function of GA 20-oxidase-likegenes of Satsuma mandarin, CuGA20ox1 andFig. 3. Southern blot analysis of CuGA20ox1 (A) and CuGA20ox2 (B). The genomic DNA (5 μg) of ‘Silverhill’ and ‘Miyagawa-wase’ Satsuma
mandarin cultivars and other citrus/trifoliate orange species/cultivars ‘Kiyomi’ tangor, Hyuganatsu, Clementine, ‘Clementine Vita’, ‘Trovita’
sweet orange, ‘Benimadoka’ and ‘Banpeiyu’ pummelo, Sudachi, Yuzu, Kunenbo, and trifoliate orange were digested with XbaI, and thenseparated on a 0.8% agarose gel. DNA bands were transferred to Hybond N+ and hybridized with a digoxigenin (DIG)-labeled CuGA20ox1(A) or CuGA20ox2 (B) cDNA coding region. Hybridization was performed in DIG Easy Hyb (Roche Diagnostics) at 42°C for 16 h followedby two washes in 0.5× SSC containing 0.1% (v/v) SDS at 68°C for 20 min. Molecular size markers are shown in kb on the left.

Hort. J. 85 (2): 128–140. 2016.

135

Fig. 4. Phylogenetic analysis of CuGA20ox1 and CuGA20ox2 together with other GA 20-oxidases. The tree was constructed by the neighborjoining (N-J) method for the deduced amino acid sequence of the GA 20-oxidase genes from apple [MdGA20ox (AB037114), MdGA20ox(MDP0000136940)], Arabidopsis [AtGA20ox1 (AT4G25420), AtGA20ox2 (AT5G51810), AtGA20ox3 (AT5G07200), AtGA20ox4(AT1G60980), AtGA20ox5 (AT1G44090A)], Carrizo citrange [CcGA20ox1 (AJ250187), CcGA20ox2 (EU834068)], Clementine[CclGA20ox1 (Ciclev10005157m), CclGA20ox2 (Ciclev10020694m)], common bean [PvGA20ox (U70530, U70531, U70532)], European
beech [FsGA20ox1 (AJ420192)], grape [VvGA20ox (DQ508817)], lettuce [LsGA20ox1 (AB012203), LsGA20ox2 (AB012204), LsGA20ox3(AB031202)], morning glory [InGA20ox1 (AB099488), InGA20ox2 (AB099489)], pea [PsGA20ox (U70471), PsGA20ox (U58830)],
Physcomitrella patens [PpGA20ox (EU262757)], poplar [PnGA20ox1 (AB089675), PaGA20ox1a (AB090238), PtPtGA20ox1 (AJ001326)],
potato [StGA20ox1 (AJ291453), StGA20ox2 (AJ291454)], Selaginella moellendorffii [SmGA20ox (EU262754)], rice [OsGA20ox1 (U50333),
OsGA20ox2 (AB469066)], Satsuma mandarin [CuGA20ox1 (LC056054), CuGA20ox2 (LC056055)], spinach [SoGA20ox (U33330)], sweetorange [CsGA20ox1 (orange1.1g016776m), CsGA20ox2 (orange1.1g041132m)], tobacco [NtGA20ox (AB012856), NsGA20ox2(AF494088)], and tomato [LeGA20ox1 (AF049898), LeGA20ox2 (AF049899), LeGA20ox3 (AF049900), SlGA20ox4 (EU675630)].

CuGA20ox2, we introduced the coding region of eachgene under the control of the cauliflower mosaic virus(CaMV) 35S promoter fused with Ω sequence (35SΩ)
into Arabidopsis (Fig. 6A). We analyzed five independent lines with 12–13 individuals (n = 12–13) in the T2
generation for phenotypes including the numbers ofdays to flowering, the numbers of rosette and cauline
leaves at flowering, and the numbers of inflorescences
and lengths of first inflorescences one month after incubation (Fig. 6B; Table 2). For the numbers of days toflowering, rosette and cauline leaves, and inflorescences, there were no significant differences between
transgenic lines with 35SΩ::CuGA20ox1 and
35SΩ::CuGA20ox2 and control plants [Wt (Col): wildtype plants; Vector/wt (Col): transgenic lines with anempty vector]. However, transgenic lines 1-1, 1-2, and1-5 of 35SΩ::CuGA20ox1 with values of 23.19, 23.00,
and 22.81 cm, respectively, and transgenic lines 2-1 and2-3 of 35SΩ::CuGA20ox2 with values of 23.96 and23.46 cm, respectively, showed statistically significant
differences from wt (Col) and vector/wt (Col) with values of 13.21 and 17.35 cm, respectively in the length of
the first inflorescences at the P value of 0.05, resulting
in longer stems than those of control plants (Fig. 6B;
Table 2).

In addition to the phenotypic analysis, the transgene
expression in five independent lines per each construct
was confirmed by qRT-PCR. From the transgenic lines
with 35SΩ::CuGA20ox1, lines 1-2 and 1-5 showed thehighest expression, followed by lines 1-1, 1-3, and 1-4
in decreasing order (Fig. 7A). From the transgenic lines
with 35SΩ::CuGA20ox2, line 2-3 showed the highest
expression, followed by lines 2-5, 2-1, 2-2, and 2-4 indecreasing order (Fig. 7B). As expected, the lines withrelatively higher expression of each transgene, such aslines 1-1, 1-2, 1-5, 2-1, and 2-3 (indicated by asterisks
in Figure 7), showed the phenotype of significantly
elongated inflorescence, although line 2-5, which expressed CuGA20ox2 at the same level as or slightly
higher than line 2-1, did not show a significant difference in the length of the first inflorescence (Fig. 7B).
Effect of CuGA20ox1 and CuGA20ox2 on the level ofgibberellins in transgenic Arabidopsis
In transgenic lines with 35SΩ::CuGA20ox1, designated as line 1 in this experiment, GA19 decreased,
whereas both GA29 and GA8 increased significantly on
the early-13-hydroxylation pathway (Fig. 8A, C, D).
GA20 tended to increase as compared with that in awild-type control (Fig. 8B). On the non-13-

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Fig. 5. Expression patterns of CuGA20ox1 (A) and CuGA20ox2(B) in various Satsuma mandarin tissues by quantitative realtime RT-PCR. The samples for (A) and (B) from left to rightare as follows: seeds, roots, stems, new leaves (NL), old leaves(OL), and nodes of two-year-old nucellar seedlings of a cultivar‘Silverhill’ in the juvenile phase; NL, OL, and shoot apices(SA) in the adult phase; flower buds (FB), young fruit (YF) in
June, juice sacs (JS) in September, November, and December,and peels (PL) in September and December in the reproductive
phase. Levels of detected amplicons were normalized by reference to CuActin (Citrus unshiu actin gene). Values are means ±SD of the results from three replicates. The primer sets used inthis experiment and the PCR conditions are described inTable 1.

hydroxylation pathway in line 1, GA24 decreased,
whereas GA34 increased significantly (Fig. 8E, H). In
transgenic lines with 35SΩ::CuGA20ox2, designated asline 2, GA20 increased significantly on the early-13hydroxylation pathway (Fig. 8B). For other 13-OH GAs
in line 2, changes in the levels of these GAs were notsignificantly different from those of the control. On the
non-13-hydroxylation pathway in line 2, GA24 decreased, whereas GA34 increased significantly as in line
1 (Fig. 8E, H). GA1 and GA4, important active GAs on
the early-13-hydroxylation and non-13-hydroxylation
pathways, respectively, could not be detected in lines 1
or 2 or in a wild-type control (data not shown).

Fig. 6. Schematic representation of transformation vectors (A) andtypical phenotypes of transgenic Arabidopsis with Satsuma
mandarin CuGA20ox1 (line1-5#7) and CuGA20ox2 (line2-2#7)
genes 24 days after incubation under long-day conditions. (B)Wt: wild-type plants; Vector: transgenic control plants with anempty vector.

Discussion
Satsuma mandarin has two GA 20-oxidase genes,
CuGA20ox1 and CuGA20ox2. The genomic sequence
for CuGA20ox1 is shorter than that for CuGA20ox2,although the coding region of cDNA for GuGA20ox1
is slightly longer than that for CuGA20ox2, as Huerta
et al. (2009) reported regarding two GA 20-oxidase
genes in Carrizo citrange (Fig. 2). The amino acid sequence deduced from CuGA20ox1 was identical to thatin Clementine and very similar to that in Carrizo
citrange and sweet orange; the amino acid sequencededuced from CuGA20ox2 was identical to those in
Clementine and sweet orange and very similar to that in
Carrizo citrange (Figs. 2B and 4B). Southern blot analysis revealed that Citrus species/cultivars and trifoliateorange have two types of GA 20-oxidase genes. Especially, the blot probed with CuGA20ox2 showedpolymorphic bands, indicating the diversity of
citrus GA20ox2 genes, although the blot probedwith CuGA20ox1 showed single bands at a similar molecular weight, indicating conserved sequences aroundthe XbaI site in citrus GA20ox1 genes. In considering

Table 2. Characteristics of transgenic lines ectopically expressing CuGA20ox1 or CuGA20ox2.
Hort. J. 85 (2): 128–140. 2016.
Table 2.

137

Characteristics of transgenic lines ectopically expressing CuGA20ox1 or CuGA20ox2.
LD conditions (16-h light/8-h dark)

Line

No. ofplantsWt (Col)
14
Vector/wt (Col)1435SΩ:CuGA20ox1/wt (T2)Line 1-1
13Line 1-2
13Line 1-3
13Line 1-4
12Line 1-5
13
35SΩ:CuGA20ox2/wt (T2)Line 2-1
13Line 2-2
13Line 2-3
12Line 2-4
12Line 2-5
12No. of days toflowering

No. of rosetteleavesNo. of cauline
leavesNo. ofinflorescences

Length of 1st infl.
(cm)

29.14 ± 1.83 a
27.54 ± 1.71 abcd

6.50 ± 0.85 a
7.00 ± 1.00 a

3.14 ± 0.86 a
3.85 ± 1.07 abc

1.00 ± 0.00 a
1.15 ± 0.38 a

13.21 ± 3.82 a
17.35 ± 3.27 ab

25.23 ± 2.24 d
26.00 ± 2.06 bcd
27.46 ± 3.57 abcd
25.75 ± 1.78 bcd
26.15 ± 2.23 abcd

6.77 ± 1.30 a
7.08 ± 0.86 a
7.77 ± 1.79 a
6.42 ± 0.90 a
5.92 ± 1.12 a

3.69 ± 0.75 abc
3.77 ± 0.73 abc
3.46 ± 0.78 ab
3.67 ± 0.89 abc
4.46 ± 0.97 bc

1.23 ± 0.44 a
1.23 ± 0.44 a
1.15 ± 0.36 a
1.33 ± 0.49 a
1.00 ± 1.00 a

23.19 ± 2.95 cd
23.00 ± 3.50 cd
18.50 ± 5.75 bc
21.63 ± 4.24 bcd
22.81 ± 4.23 cd

25.08 ± 1.44 d
26.85 ± 2.88 abcd
25.67 ± 1.56 cd
28.75 ± 2.60 abc
28.92 ± 3.65 ab

6.08 ± 0.64 a
6.77 ± 1.17 a
6.58 ± 0.90 a
5.75 ± 0.62 a
6.25 ± 1.76 a

3.92 ± 0.95 abc
3.62 ± 0.65 ab
4.75 ± 0.75 c
2.83 ± 0.83 a
3.67 ± 0.49 abc

1.15 ± 0.38 a
1.08 ± 0.28 a
1.08 ± 0.29 a
1.17 ± 0.39 a
1.08 ± 0.29 a

23.96 ± 2.45 d
19.58 ± 3.09 bcd
23.46 ± 2.62 d
16.75 ± 1.74 ab
16.71 ± 2.90 ab

Plants in the second generation (T2) and controls were grown under long-day (LD) conditions. Numbers of rosette and cauline leaves were counted
on the day floral organs became visible. Numbers of inflorescences merging from the base were counted and length of 1st inflorescences (infl.) was
measured one month after incubation. Values are means ± standard deviation (SD). Tukey’s multiple comparisons were performed to compare theeffects of the ectopic expression of the genes. Superscripts letters indicate a statistically significant difference from results with other letters andvalues in bold indicate a statistically significance from those of controls in the same column (P < 0.05).

the genealogy of Hyuganatsu it is interesting that
Hyuganatsu has two bands—an upper band that is also
seen in some mandarins and Yuzu and a lower band thatis also seen in a pummelo cultivar (‘Benimadoka’). The
deduced amino acid sequences of CuGA20ox1 andCuGA20ox2 were identical to those of Clementine, confirming evolutionally close lineage between Satsuma
mandarin and Clementine. Variation in the genome sequences of CuGA20ox2 might explain the characteristics of flower and ovary development, which vary
among species and cultivars in citrus.
CuGA20ox1 and CuGA20ox2 were differentially expressed in various tissues as shown in Figure 5.
CuGA20ox1 was expressed in almost all of the tissues
investigated, with relatively higher expression in vegetative than in reproductive tissues, whereas CuGA20ox2
was specifically expressed in flower buds just before
anthesis. These expression patterns were quite similar
to those of CcGA20ox1 and CcGA20ox2 in Carrizo
citrange (Huerta et al., 2009). The distinct expression
patterns of CuGA20ox1 and CuGA20ox1 imply that thefunction of these two genes diverged in the process ofevolution. CuGA20ox1 was expressed in seeds but
CuGA20ox2 was not, suggesting that CuGA20ox1
would regulate the basal level of active GAs in citrus
seeds. On the other hand, CuGA20ox2 was expressed
specifically in flower buds, suggesting that it would be
responsible for the level of active GAs, such as GA1,
GA19, and GA20, which are found in flower buds including ovaries at the early developmental stage or young
fruit in Satsuma mandarin (Goto et al., 1989). The specific and relatively higher expression of GuGA20ox2 inflower buds would explain why GA-like activity ishigher in Satsuma mandarin ovaries at anthesis (Takagi
et al., 1989; Talon et al., 1990b) and why Satsuma mandarin show a higher rate of fruit growth than does
Clementine at the early developmental stage aroundanthesis (Talon et al., 1990b). Gene expression of
CuGA20ox2 in reproductive oragans of various species
and cultivars remain to be investigated in detail.
Transgenic Arabidopsis plants ectopically expressing
CuGA20ox1 or CuGA20ox2 were examined to elucidate
the function of these two genes of Satsuma mandarin.
Phenotypic analysis revealed that both CuGA20ox1 andCuGA20ox2 caused elongated inflorescence, but did notaffect the timing of flowering in transgenic Arabidopsis
as compared with wild-type controls (Fig. 6; Table 2).
As expected, transgene expression in each line is positively related to the length of inflorescence, namely, thelines with relatively higher expression of CuGA20ox1
or CuGA20ox2 showed an elongated phenotype with astatistically significant difference except for line 2-5(Fig. 7). Similar phenotypes were observed in transgenic Arabidopsis and tobacco plants ectopically expressing AtGA20ox and CcGA20ox1, respectively,
which were taller and had larger inflorescences thanthose of control plants (Coles et al., 1999; Vidal et al.,2001).
CuGA20ox1 and CuGA20ox2 in transgenic
Arabidopsis functioned as CcGA20ox1 in transgenic
tobacco, therefore, the concentration of endogenous
GAs was determined by using LC-MS/MS to clear theeffect of these gene products on the GA biosynthetic
pathways in transgenic Arabidopsis. Consistent with

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N. Kotoda, S. Matsuo, I. Honda, K. Yano and T. Shimizu

Fig. 7. Expression analysis for transgenes in whole plants of transgenic Arabidopsis. (A) Gene expression of CuGA20ox1 intransgenic lines. (B) Gene expression of CuGA20ox2 in transgenic lines. Levels of detected amplicons were normalized byreference to CuActin. Values are means ± SD of the resultsfrom three replicates per line. Asterisks represent the lines thatexhibit an elongated phenotype with a statistically significant
difference from the wild-type controls shown in Table 2.

convertion of GA24, a substrate of a GA 20-oxidase, toGA9, a precursor of an active form of GA4. In addition,
an increased concentration of GA34 suggested that mostactive GA4 was converted to GA34, an inactive metabolite of GAs, during two months of growing. Likewise,on the early-13-hydroxylation pathway, ectopic expression of CuGA20ox1 significantly decreased GA19 andincreased GA29 and GA8, which were inactive metabolites of 2-hydroxylation of GA20 and GA1, respectively,
suggesting the activation of this biosynthetic pathway(Fig. 8A, C, D). Fos et al. (2000) reported that unpollinated ovaries of a pat-2 tomato mutant (Cuarenteno/
pat-2) showing natural parthenocarpy contained lessGA1 and more GA8 than those of wild type plants andalso suggeted that the content of GA8 may be a measureof the previous GA1 content because there was a correlation between the level of GA8 produced from GA20
and pea inernode elongation (Ingram et al., 1986).
CuGA20ox2 also had a tendency to activate theearly-13-hydroxylation pathway although it increased
only GA20 with a statistically significant difference(Fig. 8B). Taken together, we concluded thatCuGA20ox1 and/or CuGA20ox2 activate both theearly-13- and non-13-hydroxylation pathways, resulting
in elongated inflorescences in transgenic Arabidopsis.
CcGA20ox1 was confirmed to encode for a GA 20oxidase by utilizing the heterologous expression system
in Escherichia coli, showing activities on both GA12
and GA53 as a substrate (Vidal et al., 2003); however,
the expression product of CcGA20ox2 catalyzed only
the conversion of GA12 to putative GA9 (Huerta et al.,2009). How these two GA 20-oxidases regulate andcoordinate the GA biosynthetic pathways in citrus must
be studied to clarify more than a few physiologicalphenomena, such as flowering, fruit development andseedlessness, mediated by GA signal transduction.

Acknowledgements
the fact that Arabidopsis mainly utilizes the non-13hydroxylation pathway to accumulate active GAs suchas GA4, the concentrations of GA9, GA51, and GA34
(Fig. 8F–H) are relatively higher on the non-13hydroxylation pathway than are concentrations of GA20,
GA29, and GA8 (Fig. 8B–D), respectively, on theearly-13-hydroxylation pathway, although the concentration of GA24 (Fig. 8E) is lower than that of GA19
(Fig. 8A) in the wild-type control. Active GA1 and GA4
could not be detected in this experiment likely because
GAs were measured in two-month-old plants that wereentering the stage of senescence, although Coles et al.
(1999) detected both GA1 and GA4 in shoot tips of
Arabidopsis plants about one month after sowing. Ectopic expression of CuGA20ox1 and CuGA20ox2 significantly affected the levels of GA24 and GA34 on the
non-13-hydroxylation pathway; GA24 decreased andGA34 increased (Fig. 8E, H). This observation indicatedthat both CuGA20ox1 and CuGA20ox2 accelerated theWe thank Dr. H. Ichikawa for providing the binary
vector pSMAK193E, Dr. E. E. Hood for providing
Agrobacterium tumefaciens EHA101, and Ms. C.
Hirano and Y. Uchida for their technical assistance.

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