This article is an Advance Online Publication of the authors’ corrected proof. Note that minor changes may be made before final version publication.

The Horticulture Journal Preview
doi: 10.2503/hortj.OKD-016

The Japanese Society for
JSHS

Horticultural Science

http://www.jshs.jp/

Gibberellin 2-Oxidase Genes from Satsuma Mandarin (Citrus unshiu Marc.)
Caused Late Flowering and Dwarfism in Transgenic Arabidopsis
Nobuhiro Kotoda1*,**, Satoshi Matsuo2, Ichiro Honda3, Kanako Yano1
and Tokurou Shimizu1
1

Institute of Fruit Tree and Tea Science, NARO, Shizuoka 424-0292, Japan
Institute of Vegetable and Floriculture Science, NARO, Tsu 514-2392, Japan3
Department of Biotechnology, Maebashi Institute of Technology, Maebashi 371-0816, Japan

2

Gibberellins (GAs) are involved in many aspects of plant growth and development in higher plants. In thisstudy, we aimed to characterize Satsuma mandarin (Citrus unshiu Marc.) GA 2-oxidase genes encoding
enzymes with GA inactivation activity because the accumulation of active GAs is regulated by the balance
between their synthesis and inactivation. We showed that CuGA2ox4, CuGA2ox2/3, and CuGA2ox8 weredifferentially expressed in various tissues in Satsuma mandarin and that these genes functioned like GA 2oxidase genes in transgenic Arabidopsis. The expression pattern of CuGA2ox4 resembled that of CuGA20ox1,
which was reported previously as a Satsuma mandarin GA 20-oxidase gene, in the reproductive tissues of theadult trees in that both genes were expressed in juice sacs in November and December and peel in December,suggesting that CuGA2ox4 and CuGA20ox1 act in concert to regulate the accumulation of active GAs in fruitat the maturing stage. On the other hand, CuGA2ox2/3 and CuGA2ox8 were more highly expressed in seeds
and flower buds, respectively. Further study of GA biosynthetic genes, including GA 2-oxidase genes, would
provide insight into the mechanism of flowering, fruit development, seedlessness, biennial bearing, and peel
puffins of citrus such as Satsuma mandarin.
Key Words: fruit tree, gene expression, parthenocarpy, plant growth, seedless fruit.

Introduction
Extensive studies over many years revealed that gibberellins (GAs) are involved in many aspects of plant
growth and development, such as cell elongation, cell
division, dormancy, flowering, and fruit development inhigher plants. Researchers have also conducted biochemical and molecular studies to clarify the biosynthetic pathways of GAs in plants (Hedden and Kamiya,
1997; Hedden and Phillips, 2000; Yamaguchi, 2008).
Received; May 13, 2016. Accepted; August 1, 2016.
First Published Online in J-STAGE on September 27, 2016.
This study was supported by a Grant-in-Aid for Scientific Research(C) from the Ministry of Education, Culture, Sports, Science andTechnology (No. 24580058) and a Research Project for Molecular
Evaluation of Parthenocarpy on Horticultural Crops from NARO
(No. 210).
* Corresponding author (E-mail: koto@cc.saga-u.ac.jp; nobu_
fruits@yahoo.co.jp).
** Present address: Faculty of Agriculture, Saga University, Saga
840-8502, Japan

These studies revealed that there are 2 pathways forsynthesizing active GAs; the early-13-hydroxylation
pathway, where GAs such as GA1 and GA3, are synthesized sequentially through GA53, GA44, GA19, and GA20
after the synthesis of GA12 through some steps fromisopentenyl diphosphate and dimethylallyl diphosphate
and the non-13-hydroxylation pathway where GAs,such as GA4, are synthesized through GA15, GA24, andGA9 after the synthesis of GA12. In the biosynthetic
pathway, GA 20- and GA 3-oxidase genes accelerate
the synthesis of the active GAs, whereas GA 2-oxidase
genes inactivate the active GAs through 2βhydroxylation.
Citrus, such as Satsuma mandarin (Citrus unshiu
Marc.), is thought to use the early-13-hydroxylation
pathway because citrus accumulates 13-OH GAs suchas GA1, more abundantly than it accumulates 13-H GAssuch as GA4 (Goto et al., 1989; Poling and Maier, 1988;
Talon et al., 1990a, b; Turnbull, 1989). In addition to
the study in rice and Arabidopsis, much effort has been
devoted to understanding the physiological role of GAs© 2016 The Japanese Society for Horticultural Science (JSHS), All rights reserved.

2

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

in woody plants, including fruit trees. It has been demonstrated that in apple (Malus × domestica Borkh.) andcitrus trees, GAs function negatively in terms of flower
induction (Luckwill and Silva, 1979; Monselise, 1979;
Tromp, 1982). On the other hand, Nagao et al. (1989)
reported that GAs play a positive role in the floweringof the Japanese cedar (Cryptomeria japonica). The rosette and dwarf seedlings with internodes of less than
1 mm obtained from the selfing and crossing of trifoliate orange (Poncirus trifoliata Raf.) strains, such as the‘Hiryu’ (‘Flying Dragon’), responded to GA3 application with a significant elongation of the internodes, suggesting the importance of internal GAs for internode
elongation in citrus (Yoshida, 1994). In several dwarf
plum (Prunus salicina L.) genotypes, a markedly elevated level of a GA 2-oxidase gene (PslGA2ox) expression was responsible for the dwarfism, and PslGA2ox
could be a marker for developing size-controlling rootstocks in prunus (El-Sharkawy et al., 2012). In grapes, apractical method for producing seedless berries by applying GA3 was established as early as the late 1950s(Kishi and Tasaki, 1960).
The production of seedless fruit is also very important for breeders and growers to meet consumer demands in citriculture. Satsuma mandarins are
considered to be absolutely seedless because it has both
female and male sterility with parthenocarpy (Iwamasa,
1966; Vardi et al., 2008). Talon et al. (1990b) reported
that in Satsuma mandarin, the development of ovaries
preceded that in Clementine (Citrus clementina hort. ex
Tanaka), which shows self-incompatibility and low parthenocarpy. In addition, active GAs and their precursors
were more highly accumulated in the ovaries at aroundanthesis in Satsuma mandarin than in Clementine(Talon et al., 1990b, 1992). These results imply a relationship between the degree of seedlessness and the endogenous GAs. In recent years, GA biosynthetic genes
have been isolated and analyzed to clarify their functions in citrus (Fagoaga et al., 2007; Huerta et al., 2009;
Kotoda et al., 2016; Vidal et al., 2001, 2003). A previous study identified two GA 20-oxidase genes from
Satsuma mandarin and confirmed their function usingtransgenic Arabidopsis. Consistent with the accumulation of active GAs in the ovaries at around anthesis,
CuGA20ox2 was specifically expressed in the flower
bud just before anthesis in Satsuma mandarin (Kotoda
et al., 2016). In this study, Satsuma mandarin GA 2oxidase genes encoding enzymes with an inactivation
activity of GAs were characterized because the accumulation of active GAs is regulated by the balance between their synthesis and inactivation. Molecularcharacterization of GA biosynthetic genes, including
GA 2-oxidase genes, would be helpful in studying themechanisms of important physiological events such as
flowering and parthenocarpy in citrus.

Materials and Methods
Plant materials
Tissue samples of a Satsuma mandarin (Citrus unshiu
Marc.) ‘Silverhill’ in the adult phase (age: 43 years)
were collected from the experimental field at the Institute of Fruit Tree and Tea Science, NARO, 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.
Seeds used in this study were derived from ‘Silverhill’
Satsuma mandarin harvested in January in 2009.
Satsuma mandarins (‘Silverhill’ and ‘Miyagawa wase’),‘Kiyomi’ tangor (C. unshiu × C. sinensis Osbeck),Hyuganatsu (C. tamurana hort. ex Tanaka), Clementine,‘Clementine Vita’ (Citrus clementina hort. ex Tanaka‘Clementine Vita’), sweet orange (C. sinensis Osbeck‘Trovita’), pummelo (C. grandis Osbeck ‘Benimadoka’and ‘Banpeiyu’), Sudachi (C. sudachi hort. ex Shirai),
Yuzu (C. junos Sieb. ex Tanaka), Kunenbo (C. nobilis
Lour.), and trifoliate orange were used for Southern blot
analysis. Wild-type plants of the ecotype Columbia(Col) were used for Arabidopsis [Arabidopsis thaliana(L.) Heynh] transformation. Arabidopsis seeds werestratified for 3 to 4 days at 4°C and then grown on anagar plate containing 0.5× Murashige and Skoog (MS)medium (Wako Pure Chemicals Co. Ltd., Tokyo, Japan)
in a growth chamber at 22°C. Ten days after incubationon the agar plate, seedlings were transferred to soil andgrown in growth chambers under long day (LD) conditions (16 h light/8 h dark).
Isolation of GA 2-oxdase genes from Satsuma mandarin
To obtain genomic sequences of GA 2-oxidase genes
from Satsuma mandarin, we screened a bacterial artificial clone (BAC) library of Satsuma mandarin(‘Miyagawa wase’) and sequenced three clones named
STR00402, STR00547, and STR00577, which contained homologous genes of GA 2-oxidase. The 3 GA
2-oxidase-like genes in the clones STR00402,
STR00547, and STR00577 were designated as
CuGA2ox4 (Accession No. LC149862), CuGA2ox2/3(Accession No. LC149863), and CuGA2ox8 (Accession
No. LC149864) based on the phylogenetic analysis
described below. Leaves and ovaries of ‘Silverhill’
Satsuma mandarin were used to isolate the cDNA of
CuGA2ox4, CuGA2ox2/3, and CuGA2ox8. cDNA fromthe leaves and ovaries was amplified by polymerase
chain reaction (PCR) with the oligonucleotide primers
STR00402_F1 and STR00402_R1 for CuGA2ox4,
STR00547_F1 and STR00547_R1 for CuGA2ox2/3,
and STR00577_F1 and STR00577_R1 for CuGA2ox8,
designed based on the genomic sequences of
CuGA2ox4, CuGA2ox2/3, and CuGA2ox8, respectively,
using a taq polymerase, KOD-plus (Toyobo, Osaka,
Japan). Each amplified product was cloned into the3Hort. J. Preview

EcoRV site of the pBlueScript II SK(+) vector (Agilent
Technologies, Santa Clara, CA, USA) after phosphorylation of the products. All PCR products for isolation of
the cDNA of CuGA2ox4, CuGA2ox2/3, and CuGA2ox8
were subcloned and sequenced (ABI 3130xl DNA sequencer; Life Technologies, Carlsbad, CA, USA). The
oligonucleotide primer sets used in gene cloning arelisted in Table 1.
Construction of a phylogenetic tree
The amino acid sequences were aligned using theClustalX multiple sequence alignment program ver.
2.0.5 (Larkin et al., 2007; Thompson et al., 1997), andthe phylogenetic tree was constructed by the neighborjoining (N-J) method for the deduced amino acid sequence of the GA biosynthetic genes from Arabidopsis[AtGA2ox1 (AT1G78440), AtGA2ox2 (AT1G30040),
AtGA2ox3 (AT2G34555), AtGA2ox4 (AT1G47990),
AtGA2ox6 (AT1G02400), AtGA2ox7 (AT1G50960),
AtGA2ox8 (AT4G21200), AtGA3ox1 (AT1G15550),
AtGA3ox2 (AT1G80340), AtGA3ox3 (AT4G21690),
AtGA3ox4 (AT1G80330), AtGA20ox1 (AT4G25420),
AtGA20ox3 (AT5G07200), and AtKAO2 (AT2G32440)]
and Satsuma mandarin [CuGA20ox1 (LC056054),
CuGA2ox4 (LC149859), CuGA2ox2/3 (LC149860), andCuGA2ox8 (LC149861)]. The unrooted tree was dis-played using the NJplot program (Perrière and Gouy,
1996). The alphanumeric characters in parentheses
represent the accession numbers used in TAIR (TheArabidopsis Information Resource, https://www.
arabidopsis.org/) or GenBank/EMBL/DDBJ.
DNA extraction and hybridization analysis
Genomic DNA was isolated using a cetyltrimethylammonium bromide (CTAB)-based method as described 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).
Each blotted membrane was hybridized with digoxigenin (DIG; Roche Diagnostics, Mannheim, Germany)—labeled CuGA2ox4, CuGA2ox2/3, or CuGA2ox8 cDNA,
which was amplified by PCR with a pair of oligonucleotide primers used for gene cloning and a DIG mixture (Roche Diagnostics). Hybridization was performedin DIG Easy Hyb (Roche Diagnostics) at 42°C for 16 h,followed by 2 rinses in 2x saline sodium citrate (SSC)
containing 0.1% (w/v) sodium dodecyl sulfate (SDS)
at room temperature for 2 min and 2 washes in 0.5x
containing 0.1% (w/v) SDS at 68°C for 20 min as described by Kotoda et al. (2006). Detection was

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

Table 1.

Oligonucleotide primer sets used in cloning, quantitative real-time RT-PCR analysis, vector construction, and probe labelling.
Gene

Cloning/probe labelling
CuGA2ox4
CuGA2ox2/3
CuGA2ox8Real-time RT-PCR
CuGA2ox4(amplicon, 138 bp)
CuGA2ox2/3(amplicon, 125 bp)
CuGA2ox8(amplicon, 134 bp)
Vector construction
CuGA2ox4
CuGA2ox2/3
CuGA2ox8

PrimerOligonucleotide (5′ → 3′)

STR00402_F1
STR00402_R1
STR00547_F1
STR00547_R1
STR00577_F1
STR00577_R1

GTTCTGATATTTCATGCAATTAGATTCAGAAGATGCCTCAAAAAGAAATTAGAAGTAGTAGCC
ACTTCGACAAAAACCCTAATGTTCTTACCAGCAGAAGTCTAAACTAATCGTAGAAAACATTC

STR00402 (379 > 402)
STR00402 (491 < 516)
STR00547 (753 > 775)
STR00547 (851 < 877)
STR00577 (902 > 925)
STR00577 (1011 < 1035)

GACCCTTCAAAATTCGGGTCAGCG
AACGTCCCTGATCATCTTACTGAAGTTGCCTTGCAG GTAATGACTAATG
ATCTTTTCGCTCAAAGGTGGCCGAGACTTATTCCAGGCTTGGAGCATGAATTCTGAATCACAGTGTCGTAC

STR00402_F1-XbaI
STR00402_R1-SacI
STR00547_F1-XbaI
STR00547_R1-KpnI
STR00577_F1-XbaI
STR00577_R1-SacI

AAtctagaGTTCTGATATTTCATGCAATTA
AAgagctcGATTCAGAAGATGCCTCAAAA
AAtctagaAGAAATTAGAAGTAGTAGCC
AAggtaccACTTCGACAAAAACCCTAAT
AAtctagaGTTCTTACCAGCAGAAGTCT
AAgagctcAAACTAATCGTAGAAAACATTC

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, 62°C for 60 s for
CuGA2ox4 [STR00402 (379 > 402) and STR00402 (491 < 516)], 95°C for 10 min, followed by 40 cycles of at 95°C for 15 s, 60°C for 60 s for
CuGA2ox2/3 [STR00547 (753 > 775) and STR00547 (851 < 877)] and CuGA2ox8 [STR00577 (902 > 925) and STR00577 (1011 < 1035)]. Charactersin lowercase indicate the site of restriction enzymes used for vector construction. Oligonucleotide primer sequences and PCR conditions for CuActin
and AtTUB4 are described in Kotoda et al. (2016).

4

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

performed according to the manufacturer’s protocol(Roche Diagnostics). Chemiluminescent signals werevisualized using the NightOWL image analyzer(Berthold Technologies, Bad Wildbad, Germany). The
oligonucleotide primer sets used for labelling are listed
in Table 1.
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 (derived from ‘Silverhill’ Satsuma mandarin harvested in January) and roots (whole roots
including lateral ones), stems, new and old leaves, andnodes of 2-year-old juvenile nucellar seedlings derived
from ‘Silverhill’ Satsuma mandarin, as well as fromnew and old leaves, shoot apexes in August, flower
buds (just before anthesis), young fruit in June, juice
sacs (in September, November, and December), andpeel (in September and December) of adult ‘Silverhill’
Satsuma mandarin trees. For transgene expression analysis, whole plants of transgenic Arabidopsis in the second generation (T2) and controls were collected 2
months after incubation, and total RNA was extracted
from 2 individuals per line by using a CTAB-based
method. 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 usinga QuantiTect Reverse Transcription Kit (Qiagen). Subsequent PCR reactions were performed with 1 μL of thefirst-strand cDNA as a template in a total volume of
12.5 μL using the ABI PRISM® 7000 (LifeTechnologies). Transcripts of CuGA2ox4, CuGA2ox2/3,
CuGA2ox8, CuActin (for reference, Phytozome
Transcript Name Ciclev10025866m), and an
Arabidopsis β tubulin gene (AtTUB4) were identified
with the specific primers. The oligonucleotide primer
sets used in the quantitative real-time RT-PCR (qRTPCR) analysis and the PCR conditions are described inTable 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 (technical replicates) and transcript levels were normalized against
each internal control.
Construction of the transformation vector
To construct vectors for the constitutive expressionof CuGA2ox4, CuGA2ox2/3, and CuGA2ox8, the coding region of each gene was amplified by PCR withpairs of oligonucleotide primers STR00402_F1-XbaIandSTR00402_R1-SacI
for
CuGA2ox4,
STR00547_F1-XbaI and STR00547_R1-KpnI for
CuGA2ox4,and
STR00577_F1-XbaIandSTR00577_R1-SacI for CuGA2ox8. Each amplified
PCR product was subsequently digested with XbaI/SacI
or XbaI/KpnI and then cloned into the correspondingsite of the modified pSMAK193E (35SΩ/pSMAK193E;
described in Kotoda et al., 2010), a kanamycin-resistantbinary vector, to be placed between the 35SΩ and 3' regions of the Arabidopsis rbcS-2B gene (TrbcS).
Arabidopsis transformation
An Agrobacterium tumefaciens strain EHA101 harboring a binary vector with 35SΩ::CuGA2ox4,35SΩ::CuGA2ox2/3, or 35SΩ::CuGA2ox8 was used totransform Arabidopsis thaliana (Col) plants using thefloral-dip method (Clough and Bent, 1998). After 2 to 5
adult leaves had developed on the agar plate with kanamycin in a growth chamber (Biotron; Nippon Medical
and Chemical Instruments Co., Ltd., Tokyo, Japan) set
at 22°C under LD conditions (16-h photoperiod; coolwhite fluorescent light, 50 μmol·m−2·s−1), kanamycinresistant transformants were transplanted from the plate
to moistened potting soil composed of vermiculite andperlite (1:1) and further grown in the growth chamber
under the same conditions. Morphological and expression analyses were performed on the second generation(T2). For morphological analysis, the number of days toflowering and the number of rosette leaves at flowering
were counted and the length of the 1st inflorescences(infl.) 1 month after the start of incubation was measured. Whole plants of T2 and controls were collected
2 months after the start of incubation in the growthchamber for qRT-PCR analysis.
Statistical analysis
Data on the number of days to flowering, the numberof rosette leaves, and the length of the 1st inflorescenceshown in Table 2 were analyzed using one-way
ANOVA, and the differences were contrasted using
Tukey’s multiple comparison test. All statistical analyses were performed at a significance level of P < 0.05
using R-3.2.0 (R Core Team, 2015).

Results
Isolation of CuGA2ox4, CuGA2ox2/3, and CuGA2ox8
cDNA from Satsuma mandarin
To investigate the function of GA 2-oxidase genes in
Satsuma mandarin, we isolated the cDNA of thesegenes from Satsuma mandarin ‘Silverhill’ by PCR witholigonucleotide primer sets based on the genomic sequences of GA2ox4-, GA2ox2/3-, and GA2ox8-likegenes, which were cloned from a Satsuma mandarin(‘Miyagawa wase’) BAC library. The coding sequencesof Citrus unshiu GA2ox4-like (CuGA2ox4; Accession
No. LC149859), Citrus unshiu GA2ox2/3-like(CuGA2ox2/3; Accession No. LC149860), and Citrus
unshiuGA2ox8-like(CuGA2ox8;
Accession
No. LC149861) cDNA were 1005, 1038, and 1089 bp
long including a stop codon and encoded 334, 345, and
362 amino acids, respectively. For the genomic clonesof CuGA2ox4, CuGA2ox2/3, and CuGA2ox8, sequences
including the coding region of each gene and the exon-

Table 2. Characteristics of transgenic lines ectopically expressing CuGA2ox4, CuGA2ox2/3, or CuGA2ox8.
Hort. J. Preview

5

Table 2. Characteristics of transgenic lines ectopically expressing CuGA2ox4, CuGA2ox2/3, or CuGA2ox8.
Line
Controls
Wt (Col)
Vector/wt (Col)35SΩ: CuGA2ox4/wt (T2)
#7
#8
#9
#11
35SΩ: CuGA2ox2/3/wt (T2)
#15
#16
#17
#19
35SΩ: CuGA2ox8/wt (T2)
#15
#16
#17
#20

LD conditions (16 h light/8 h dark)
No. of plants

Days to flowering

No. of rosette leaves

Length of the 1st infl. (cm)12
10

28.17 ± 1.27ab
27.70 ± 1.77a

6.58 ± 0.79a
7.10 ± 1.20ab

16.38 ± 3.84a
16.60 ± 2.86a

13
13
13
12

27.15 ± 1.95a
32.46 ± 3.38ab
33.62 ± 3.01bc
34.92 ± 3.40d

7.92 ± 1.55ab
9.31 ± 1.38bc
8.85 ± 1.41ab
9.50 ± 2.58bc

15.62 ± 3.28a6.15 ± 3.61bc
5.69 ± 1.84cd
5.29 ± 2.35cd

13
13
13
12

41.54 ± 5.25e
36.46 ± 4.52d
29.92 ± 2.75ab
45.42 ± 1.38e

11.62 ± 1.39c
9.85 ± 1.86c
9.15 ± 1.99b
11.17 ± 2.29c

0.23 ± 0.83d
3.19 ± 5.20cd
12.69 ± 4.78ab
0.00 ± 0.00d

121335.50 ± 4.38d
33.77 ± 3.49c

9.92 ± 1.16c
9.46 ± 0.88bc

1.00 ± 1.26d
1.38 ± 1.37cd

12
12

34.58 ± 5.07c
39.25 ± 4.14d

9.58 ± 1.44bc
10.08 ± 1.08c

3.17 ± 3.64cd
0.00 ± 0.00d

Plants in the second generation (T2) and controls were grown under long-day (LD) conditions. Numbers of rosette leaves were counted on the day
floral organs became visible. The length of the 1st inflorescences was measured 1 month after the start of incubation in a growth chamber. Values
are means ± standard deviation (SD). Tukey’s multiple comparisons were performed to compare the effects of the ectopic expression of the genes.
Superscripts letters indicate a statistically significant difference from results with other letters, and values in bold indicate a statistically significant
difference from those of a control plant (vector/wt) in the same column (P < 0.05).

intron structure were identified (Fig. 1A). To examine
the evolutionary relationships among GA biosynthetic
genes such as GA 2-, 3-, and 20-oxidase genes, we
performed phylogenetic analysis of the amino acid sequences corresponding to these genes from Arabidopsis
and Satsuma mandarin. N-J distance analysis showedthat CuGA2ox4, CuGA2ox2/3, and CuGA2ox8 weregrouped closely with AtGA2ox4, AtGA2ox2/3, andAtGA2ox8, respectively (Fig. 1B). Southern blots ofthe genomic DNA of 12 Citrus species/cultivars and atrifoliate orange standard strain digested with XbaI
hybridized with the DIG-labeled cDNA of CuGA2ox4,
CuGA2ox2/3, and CuGA2ox8 showed polymorphic
bands for CuGA2ox4 and CuGA2ox8 (Fig. 2A, C) and asingle band of about 9.4 kb for CuGA2ox2/3 (Fig. 2B).
Organ-specific expression patterns of CuGA2ox4,
CuGA2ox2/3, and CuGA2ox8
To clarify the expression patterns of CuGA2ox4,
CuGA2ox2/3, and CuGA2ox8, qRT-PCR was performedon various tissues in Satsuma mandarin in the juvenile
and adult phases using oligonucleotide primers specificto each gene (Table 1). The transcript of CuGA2ox4 accumulated predominantly in reproductive tissues, suchas juice sacs (JS) in November and December and peel(PL) in December, but only faintly in roots, seeds, andflower buds (FB) (Fig. 3A). In contrast, the transcriptlevel of CuGA2ox2/3 was extremely low comparedwith those of the other 2 genes and accumulated mainly
in seeds from mature fruit and vegetative tissues, suchas roots, new leaves (NL), old leaves (OL), and nodesof seedlings in the juvenile phase, and shoot apices(SA) in the adult phase. CuGA2ox2/3 was also expressed in flower buds and juice sacs in November(Fig. 3B). The transcript of CuGA2ox8 accumulated
mainly in new leaves and flower buds in the adult phaseand stems in the juvenile phase, with low expression in
all tissues except seeds, roots, and juice sacs inDecember.
Phenotypes of transgenic Arabidopsis ectopically expressing CuGA2ox4, CuGA2ox2/3, or CuGA2ox8
To elucidate the function of GA 2-oxidase-like genes
of Satsuma mandarin, CuGA2ox4, CuGA2ox2/3, orCuGA2ox8, we introduced the coding region of eachgene under the control of the cauliflower mosaic virus(CaMV) 35S promoter fused with an Ω sequence(35SΩ) into Arabidopsis (Fig. 4A). We analyzed four
independent lines with 12–13 individuals (n = 12–13)
in the T2 generation for phenotypes, including the number of days to flowering, the number of rosette leaves atflowering, and the length of the 1st infl. 1 month after
the start of incubation in a growth chamber (Fig. 4;
Table 2). For the number of days to flowering and thelength of the 1st infl. of plants, there were significant
differences between the transgenic lines with

6

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

Fig. 1. (A) Schematic representation of the genomic organization and (B) phylogenetic analysis of CuGA2ox4, CuGA2ox2/3, and CuGA2ox8,
together with other GA biosynthetic genes. The tree was constructed by the neighbor-joining (N-J) method for the deduced amino acid sequence of the genes from Arabidopsis [AtGA2ox1 (AT1G78440), AtGA2ox2 (AT1G30040), AtGA2ox3 (AT2G34555), AtGA2ox4(AT1G47990), AtGA2ox6 (AT1G02400), AtGA2ox7 (AT1G50960), AtGA2ox8 (AT4G21200), AtGA3ox1 (AT1G15550), AtGA3ox2(AT1G80340), AtGA3ox3 (AT4G21690), AtGA3ox4 (AT1G80330), AtGA20ox1 (AT4G25420), AtGA20ox3 (AT5G07200), and AtKAO2(AT2G32440)] and Satsuma mandarin [CuGA20ox1 (LC056054), CuGA2ox4 (LC149859), CuGA2ox2/3 (LC149860), and CuGA2ox8(LC149861)]. The alphanumeric characters in parentheses represent the accession numbers in TAIR or GenBank/EMBL/DDBJ. Black boxes
represent exons and lines represent introns. Vertical bars with x represent the site of the restriction enzyme XbaI. The scale bar on the map of
the genomic organization represents approximately 1 kb.

35SΩ::CuGA2ox4, 35SΩ::CuGA2ox2/3, and 35SΩ::CuGA2ox8 and the control plants [Vector/wt(Col):transgenic lines with an empty vector]. A wild-type
Arabidopsis plants (wt) was also included in the experiment for reference. The transgenic lines of35SΩ::CuGA2ox2/3 and 35SΩ::CuGA2ox8 showed alate-flowering phenotype and shorter inflorescencescompared with the control plants. Notably, all 4 lines of35SΩ::CuGA2ox8 showed statistically siginificant differences from the control plant in days to floweirng andplant height 1 month after the start of incubation in thegrowth chamber. The transgenic lines with
35SΩ::CuGA2ox4 showed a significant late flowering
for line 9 and 11 (Table 2). As for the number of rosetteleaves, on the other hand, there were significant differences in 35SΩ::CuGA2ox2/3 and 35SΩ::CuGA2ox8 ascompared with the control plants. Three transgenic linesof 35SΩ::CuGA2ox4 showed significant shorter inflorescences than the control plants, but the flowering timeof one of them and the number of rosette leaves of all
three lines did not differ from those of the control. The
typical appearance of each transgenic line and the con-trol plants 24 days after incubation is shown in Figure
4B. The transgenic lines with 35SΩ::CuGA2ox4,35SΩ::CuGA2ox2/3, and 35SΩ::CuGA2ox8 had ashorter inflorescence and a tendency to flower later thanthe control plant, though to varying degrees. The appearance of inflorescence in each line looked similar tothat of the control except for the 1st infl. length(Fig. 4C–F), but there were some defects in the floral
oragans of the transgenic lines (Fig. 4H–J). In line 8#4of CuGA2ox4, for example, the stamens were degenerated (Fig. 4H), while, in line 15#5 of 35SΩ::CuGA2ox2/3, aberrant forms of petals and stamens
were observed (Fig. 4I). In line 15#3 of
35SΩ::CuGA2ox8, sepals and petals were slightly degenerated (Fig. 4J). Typically, the transgenic lines with
35SΩ::CuGA2ox2/3 and 35SΩ::CuGA2ox8 showed astrong phenotype of dwarfism and those with
35SΩ::CuGA2ox4 showed a weak phenotype (Fig. 4K–
M).

Hort. J. Preview

7Fig. 2. Southern blot analysis of CuGA2ox4 (A), CuGA2ox2/3 (B), and CuGA2ox8 (C). 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, weredigested with XbaI and then separated on a 0.8% agarose gel. Molecular size markers are shown in kb on the left.

Expression analysis for transgenes in whole plants oftransgenic Arabidopsis
In addition to the phenotypic analysis, the transgene
expression in four independent lines per construct was
confirmed by qRT-PCR. Of the transgenic lines with
35SΩ::CuGA2ox4, lines 9 and 11 showed higher expression, followed by lines 8 and 7, in decreasing order(Fig. 5A). Of the transgenic lines with CuGA2ox2/3,
line 16 showed the highest expression on average, fol-

lowed by lines 17, 15, and 19, in decreasing order(Fig. 5B). The transgenic lines with CuGA2ox8 showedthat lines 15 and 16 had the highest expression on average, followed by lines 17 and 20, in decreasing order(Fig. 5C). The lines with a higher expression of eachtransgene (over five times the expression of AtTUB4),
such as the lines indicated by asterisks in Figure 5,
showed a phenotype of significantly late flowering
and/or short inflorescence, although line 17 of

8

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

A
35S

CuGA2ox-like gene

Ω

B

TrbcS

24d

Wt

C

Vector/wt

26d

Vector/wt

G

Vector/wt

K

44d

D

H

CuGA2ox4

E

39d

CuGA2ox4 11#2

I

CuGA2ox4 8#4

L

CuGA2ox2/3

43d

CuGA2ox2/3 15#5

CuGA2ox2/3 15#5

43d

CuGA2ox8

F 41d

J

CuGA2ox8 20#1

CuGA2ox8 15#3

M

43d

CuGA2ox2/3 15#5

Vector/wt

Fig. 3. Expression patterns of CuGA2ox4 (A), CuGA2ox2/3 (B),and CuGA2ox8 (C) in various Satsuma mandarin tissues analyzed by quantitative real-time RT-PCR. The samples for (A)and (B), from left to right, are as follows: seeds of ‘Silverhill’
Satsuma mandarin harvested in January, roots, stems, newleaves (NL), old leaves (OL), and nodes of 2-year-old nucellar
seedlings of the cultivar ‘Silverhill’ in the juvenile phase; NL,
OL, and shoot apices (SA) in the adult phase; and flower buds
(FB), young fruit (YF) in June, juice sacs (JS) in September,
November, and December, and peel (PL) in September andDecember 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 threetechnical replicates. The primer sets used in this experimentand the PCR conditions are described in Table 1.

CuGA2ox4 8#4Vector/wt

Vector/wt

CuGA2ox8 20#1

Fig. 4. (A) Schematic representation of transformation vectors. (B)Typical phenotypes of transgenic Arabidopsis with Satsuma
mandarin CuGA2ox4 (line 8), CuGA2ox2/3 (line 19), andCuGA2ox8 (line 20) 24 days (24d) after the start of incubation
in a growth chamber under long-day conditions. (C–F) The appearance of the inflorescence of the control (C, 26d) and thetransgenic lines with CuGA2ox4 (D, 39d), CuGA2ox2/3 (E,43d), and CuGA2ox8 (F, 41d). (G–J) A close-up view of theflower of the control (G) and the transgenic lines with
CuGA2ox4 (H), CuGA2ox2/3 (I), and CuGA2ox8 (J). (K–L)
The appearance of the whole plant of the control (vector/wt)
and transgenic lines with CuGA2ox4 (K, 44d), CuGA2ox2/3 (L,43d), and CuGA2ox8 (M, 43d). Wt: wild-type plants; vector/wt:
transgenic control plants with an empty vector. White bars in
the upper right corner of the panels represent 5 mm. Numberswith ‘d’ in the upper center of the panels represent the days
after the start of the incubation in a growth chamber.

CuGA2ox2/3, which expressed CuGA2ox2/3 at a higher
level than line 19, did not show any significant differences in the number of days to flowering, the number ofrosette leaves, or the length of the first inflorescence(Fig. 5B).

Discussion
We cloned 3 GA 2-oxidase-like genes, CuGA2ox4,

Hort. J. Preview

9Fig. 5. Expression analysis for transgenes in whole plants of transgenic Arabidopsis. Gene expression of CuGA2ox4 (A), CuGA2ox2/3 (B), andCuGA2ox8 (C) in transgenic lines collected 2 months after the start of incubation in a growth chamber. Levels of detected amplicons werenormalized by reference to AtTUB4. Values are means ± SD of the results from three technical replicates per line. Asterisks represent thetransgenic lines that exhibit a late flowering and/or a short inflorescence phenotype with a statistically significant difference from the control
shown in Table 2.

CuGA2ox2/3, and CuGA2ox8, from Satsuma mandarin.
Phylogenetic analysis using putative amino acid sequences revealed that CuGA2ox4, CuGA2ox2/3, andCuGA2ox8
weregroupedwith
AtGA2ox4,
AtGA2ox2/3, and AtGA2ox8, respectively (Fig. 1B).
The exon-intron structures of CuGA2ox4 andCuGA2ox2/3, each of which has three exons, were comparable to those of AtGA2ox4 and AtGA2ox2/3 inArabidopsis genome. However, CuGA2ox8 consisted offour exons, whereas AtGA2ox8 consists of three exons(Fig. 1B). Southern blot analysis revealed thatCuGA2ox4, CuGA2ox2/3, and CuGA2ox8 genes would
be single copy genes and that there would be some variations in the non-coding sequences of CuGA2ox4 andCuGA2ox8 genes. The blot probed with CuGA2ox4 andCuGA2ox8 showed polymorphic bands, indicating diversity around the XbaI site in citrus CuGA2ox4 andCuGA2ox8 genes. There were 2 bands found for
Satsuma mandarin (‘Silverhill’ and ‘Miyagawa wase’)in both blots because two sites of XbaI were present in
the first intron and one site was present right after thethird exon for CuGA2ox4, and only one site of XbaI
was present in the first intron for CuGA2ox8 (Fig. 1A).
The reason the blot probed with CuGA2ox2/3 showed asingle band is that the CuGA2ox2/3 gene has shorter in-trons than the others do. Considering that the genealogyof Satsuma mandarin and Kunenbo may be related, it isinteresting that the band patterns in the blot probed with
CuGA2ox4 and CuGA2ox8 were purely coincidental(Fig. 2A, C).
CuGA2ox4, CuGA2ox2/3, and CuGA2ox8 were differentially expressed in various tissues, as shown in
Figure 3. CuGA2ox4 was mainly expressed in the fruitparts, such as juice sacs and peel, and CuGA2ox8 was
expressed in the new leaves and flower buds of adulttrees and in the stems of seedlings in the juvenile phase,with lower expression in other tissues. The expression
pattern of CuGA2ox4 resembled that of CuGA20ox1 in
the reproductive tissues of the adult trees in that bothgenes were expressed in juice sacs in November andDecember and in peel in December (Fig. 3A; Kotoda
et al., 2016), suggesting that CuGA2ox4 andCuGA20ox1 act in concert to regulate the accumulationof active GAs in fruit at the maturing stage. Because
these genes are expressed in both juice sacs and peel,
they might be involved in the occurrence of peel puffins
to some extent. As reported earlier, CuGA20ox1 andCuGA20ox2 were independently expressed in the newleaves and flower buds, respectively, whereas
CuGA2ox8 was highly expressed in both tissues

10

N. Kotoda, S. Matsuo, I. Honda, K. Yano and T. Shimizu(Fig. 3C; Kotoda et al., 2016). These results suggestthat CuGA2ox8 and CuGA20ox1 act in new leaves,
whereas CuGA2ox8 and CuGA20ox2 act in flower buds
to control the level of active GAs. To clarify the processof the fruit development of citrus such as Satsuma mandarin at the early stage, the expression and function of
CuGA2ox8 and CuGA20ox2 in the ovaries around anthesis should be studied in the future. On the other
hand, the expression level of CuGA2ox2/3 was lower
than that of the other two genes, although it was mainly
expressed in seeds and in the roots of seedlings, suggesting a lower contribution of CuGA2ox2/3 to the development of organs/tissues except for seeds and theroots of seedlings (Fig. 3B).
Transgenic Arabidopsis plants ectopically expressing
CuGA2ox4, CuGA2ox2/3, or CuGA2ox8 were examined to elucidate the function of these 3 genes in
Satsuma mandarin. Phenotypic analysis revealed thatCuGA2ox4, CuGA2ox2/3, and CuGA2ox8 caused reduced inflorescence in transgenic Arabidopsis as compared with the control (Fig. 4; Table 2). As expected,
the lines with higher expression of CuGA2ox4,
CuGA2ox2/3, or CuGA2ox8 showed a late-flowering
and/or a short inflorescence phenotype, with a statistically significant difference except for line 17 of 35S::
CuGA2ox2/3 (Fig. 5). The transgenic plants with
CuGA2ox2/3 or CuGA2ox8 showed a severe dwarf phenotype (Fig. 4L, M). For the timing of flowering, all 4lines of CuGA2ox8 and 3 of 4 lines of CuGA2ox2/3
showed significantly delayed flowering as compared tothe control plants, although only 2 lines of CuGA2ox4
flowered significantly later than the control. These results demonstrated that CuGA2ox2/3 and CuGA2ox8
functioned like GA 2-oxidase genes and worked more
effectively than CuGA2ox4. Similarly, dwarf and lateflowering phenotypes were reported in transgenic
Arabidopsis and tobacco plants ectopically expressing
AtGA2ox7 and AtGA2ox8 (Schomburg et al., 2003). In
the hybrid aspen, dwarf mutant trees (stumpy) were obtained with an increased expression of PtaGA2ox1, a
GA 2-oxidase gene from the same family as AtGA2ox4,by activation tagging (Busov et al., 2003). In addition,transgenic aspen plants tissue-specifically expressing
AtGA2ox2 or AtGA2ox8 exhibited a lower growth rate
than the wild-type plants, resulting in reduced shootelongation (Eriksson et al., 2015; Mauriat et al., 2011).
In the transgenic lines showing a severe dwarf phenotype, some defects were observed in the floral organs
such as the petals and stamens (Fig. 4H–J). Similar defects in floral organs were reported in the transgenic
Arabidopsis ectopically expressing PslGA2ox, a plumGA 2-oxidase gene classified in the same group as
CuGA2ox2/3 (El-Sharkawy et al., 2012). These aberrant
organs would be produced due to a disturbance in the
distribution and/or concentration of GAs caused by theectopic expression of GA 2-oxidase genes introduced.
Based on our results and reports on other species,

CuGA2ox4, CuGA2ox2/3, and CuGA2ox8 would function as GA 2-oxidase genes and reduce the concentration of active GAs in specific tissues in Satsuma
mandarin. In citriculture, it is also important to study
the phenomina called biennial bearing because it affects
the annual production of fruit, causing annual pricefluctuations. In apple, 3 quantitative trait loci (QTLs)
for biennial bearing were detected, and MdGA2ox2b (anapple homolog of CuGA2ox2/3) and MdGA2ox8a (anapple homolog of CuGA2ox8) were located near a biennial bearing QTL on the south end of linkage group 10(Guitton et al., 2012). Because CuGA2ox2/3 andCuGA2ox8 were relatively highly expressed in seeds
and flower buds, respectively, CuGA2ox2/3 could be involved in biennial bearing and CuGA2ox8 might be involved in fruit set in Satsuma mandarin.
We conclude that CuGA2ox4, CuGA2ox2/3, andCuGA2ox8 were differentially expressed in various tissues in Satsuma mandarin and that these genes functioned like GA 2-oxidase genes in transgenic
Arabidopsis. Further study of GA biosynthetic genes including GA 2-oxidase genes will provide insight into
the mechanism of flowering, fruit development, seedlessness, biennial bearing, and the peel puffins of citrus
such as Satsuma mandarin.

Acknowledgments
We thank Dr. H. Ichikawa for providing the binary
vector pSMAK193E, Dr. E. E. Hood for providing
Agrobacterium tumefaciens EHA101, and Ms. C.
Hirano and Ms. Y. Uchida for their technical assistance.

Literature Cited
Busov, V. B., R. Meilan, D. W. Pearce, C. Ma, S. B. Rood and
S. H. Strauss. 2003. Activation tagging of a dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that
regulates tree stature. Plant Physiol. 132: 1283–1291.
Clough, S. J. and A. F. Bent. 1998. Floral dip: a simplified
method for Agrobacterium-mediated transformation of
Arabidopsis thaliana. Plant J. 16: 735–743.
El-Sharkawy, I., W. El Kayal, D. Prasath, H. Fernández, M.
Bouzayen, A. M. Svircev and S. Jayasankar. 2012. Identification and genetic characterization of a gibberellin 2-oxidase
gene that controls tree stature and reproductive growth inplum. J. Exp. Bot. 63: 1225–1239.
Eriksson, M. E., D. Hoffman, M. Kaduk, M. Mauriat and T.
Moritz. 2015. Transgenic hybrid aspen trees with increased
gibberellin (GA) concentrations suggest that GA acts in parallel with FLOWERING LOCUS T2 to control shoot elongation. New Phytologist 205: 1288–1295.
Fagoaga, C., F. R. Tadeo, D. J. Iglesias, L. Huerta, T. Lliso, A. M.
Vidal, M. Talon, L. Navarro, J. L. García-Martínez and L.
Peña. 2007. Engineering of gibberellin levels in citrus bysense and antisense overexpression of a GA 20-oxidase genemodifies plant architecture. J. Exp. Bot. 58: 1407–1420.
Goto, A., H. Yamane, N. Takahashi and K. Hirose. 1989. Identification of nine gibberellins from young fruit of Satsuma mandarin (Citrus unshiu Marc.). Agric. Biol. Chem. 53: 2817–
2818.
Guitton, B., J.-J. Kelner, R. Velasco, S. E. Gardiner, D. ChagneHort. J. Preview

and E. Costes. 2012. Genetic control of biennial bearing inapple. J. Exp. Bot. 63: 131–149.
Hedden, P. and Y. Kamiya. 1997. Gibberellin biosynthesis: enzymes, genes, and their regulation. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 48: 431–460.
Hedden, P. and A. L. Phillips. 2000. Gibberellin metabolism: new
insights revealed by the genes. Trends Plant Sci. 5: 523–530.
Huerta, L., A. Garcia-Lor and J. L. Garcia-Martinez. 2009. Characterization of gibberellin 20-oxidases in the citrus hybrid
Carrizo citrange. Tree Physiol. 29: 569–577.
Ikoma, Y., M. Yano, K. Ogawa, T. Yoshioka, Z. C. Xu, S. Hisada,
M. Omura and T. Moriguchi. 1996. Isolation and evaluationof RNA from polysaccharide-rich tissues in fruit for quality
by cDNA library construction and RT-PCR. J. Japan. Soc.
Hort. Sci. 64: 809–814.
Iwamasa, M. 1966. Studies on the sterility in genus Citrus withspecial reference to the seedlessness. Bull. Hort. Res. Sta.
Japan Ser. (B) 6: 1–81.
Kishi, T. and M. Tasaki. 1960. Budou ni taisuru Gibberellin riyou
shiken [Dai ippou] Delaware ni tsuite. Agric. Hortic.
(Nogyo-oyobi-Engei) 35: 381–384 (In Japanese).
Kotoda, N., H. Hayashi, M. Suzuki, M. Igarashi, Y. Hatsuyama,
S. Kidou, T. Igasaki, M. Nishiguchi, K. Yano, T. Shimizu, S.
Takahashi, H. Iwanami, S. Moriya and K. Abe. 2010.
Molecular characterization of FLOWERING LOCUS T-likegenes of apple (Malus × domestica Borkh.). Plant Cell
Physiol. 51: 561–575.
Kotoda, N., H. Iwanami, S. Takahashi and K. Abe. 2006. Antisense expression of MdTFL1, a TFL1-like gene, reduces thejuvenile phase in apple. J. Amer. Soc. Hort. Sci. 131: 74–81.
Kotoda, N., S. Matsuo, I. Honda, K. Yano and T. Shimizu. 2016.
Isolation and functional analysis of two gibberellin 20oxidase genes from Satsuma mandarin (Citrus unshiu
Marc.). Hort. J. 85: 128–140.
Kotoda, N., M. Wada, S. Kusaba, Y. Kano-Murakami, T. Masuda
and J. Soejima. 2002. Overexpression of MdMADS5, an
APETALA1-like gene of apple, causes early flowering intransgenic Arabidopsis. Plant Sci. 162: 679–687.
Larkin, M. A., G. Blackshields, N. P. Brown, R. Chenna, P. A.
McGettigan, H. McWilliam, F. Valentin, I. M. Wallace, A.
Wilm, R. Lopez, J. D. Thompson, T. J. Gibson and D. G.
Higgins. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.
Luckwill, L. C. and J. M. Silva. 1979. The effects of daminozide
and gibberellic acid on flower initiation, growth and fruitingof apple cv Golden Delicious. J. Hort. Sci. 54: 217–223.
Mauriat, M., L. G. Sandberg and T. Moritz. 2011. Proper gibberellin localization in vascular tissue is required to control
auxin-dependent leaf development and bud outgrowth in hybrid aspen. Plant J. 67: 805–816.
Monselise, S. P. 1979. The use of growth regulators in citriculture; A review. Sci. Hortic. 11: 151–162.

11

Nagao, A., S. Sasaki and R. P. Pharis. 1989. Cryptomeria
japonica. p. 247–269. In: A. H. Halevy (ed.) Handbook offlowering. vol VI. CRC, Boca Raton, Fla.
Perrière, G. and M. Gouy. 1996. WWW-Query: An on-line retrieval system for biological sequence banks. Biochimie 78:
364–369.
Poling, S. M. and V. P. Maier. 1988. Identification of endogenous
gibberellins in Navel orange shoots. Plant Physiol. 88: 639–
642.
R Core Team. 2015. R: A language and environment for statistical computing. R Foundation for Statistical Computing,
Vienna, Austria. <http://www.R-project.org/>.
Schomburg, F. M., C. M. Bizzell, D. J. Lee, J. A. D. Zeevaart andR. M. Amasino. 2003. Overexpression of a novel class ofgibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants. Plant Cell 15: 151–163.
Talon, M., P. Hedden and E. Primo-Millo. 1990a. Gibberellins in
Citrus sinensis: A comparison between seeded and seedlessvarieties. J. Plant Growth Regul. 9: 201–206.
Talon, M., L. Zacarias and E. Primo-Millo. 1990b. Hormonalchanges associated with fruit set and development in mandarins differing in their parthenocarpic ability. Physiol.
Plant. 79: 400–406.
Talon, M., L. Zacarias and E. Primo-Millo. 1992. Gibberellins
and parthenocarpic ability in developing ovaries of seedlessmandarins. Plant Physiol. 99: 1575–1581.
Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin and
D. G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 25: 4876–4882.
Tromp, J. 1982. Flower-bud formation in apple as affected byvarious gibberellins. J. Hort. Sci. 57: 277–282.
Turnbull, C. G. N. 1989. Identification and quantitative analysisof gibberellins in Citrus. J. Plant Growth Regul. 8: 273–282.
Vardi, A., I. Levin and M. Carmi. 2008. Induction of seedlessness
in citrus: From classical techniques to emerging biotechnological approaches. J. Amer. Soc. Hort. Sci. 133: 117–126.
Vidal, A. M., W. Ben-Cheikh, M. Talon and J. L. GarcíaMartínez. 2003. Regulation of gibberellin 20-oxidase gene
expression and gibberellin content in citrus by temperatureand citrus exocortis viroid. Planta 217: 442–448.
Vidal, A. M., C. Gisbert, M. Talon, E. Primo-Millo and I. LopezDiaz. 2001. The ectopic expression of a citrus gibberellin
20-oxidase enhances the non-13-hydroxylation pathway ofgibberellin biosynthesis and induces an extremely elongatedphenotype in tobacco. Physiol. Plant. 112: 251–260.
Yamaguchi, S. 2008. Gibberellin metabolism and its regulation.
Annu. Rev. Plant Biol. 59: 225–251.
Yoshida, T. 1994. Dwarfism in trifoliate orange (Poncirus
trifoliata Raf.), its inheritance and interaction with GA3. J.
Japan. Soc. Hort. Sci. 63: 23–30.