ORIGINAL ARTICLES
Isolation and Functional Analysis of Two Gibberellin 20-Oxidase Genes from Satsuma Mandarin (Citrus unshiu Marc.)
2016 Volume 85 Issue 2 Pages 128-140
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2016 Volume 85 Issue 2 Pages 128-140
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 expressed in 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 that function of these two genes diverged in the process of evolution. The specific and relatively higher expression of 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 the timing 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 and CuGA20ox2 accelerated the conversion of GA24, a substrate of a GA 20-oxidase, to GA9, a precursor of an active form of GA4. Likewise, on the early-13-hydroxylation pathway, ectopic expression of CuGA20ox1 significantly decreased GA19 and increased GA29 and GA8, inactive metabolites of 2-hydroxylation of GA20 and GA1, respectively, suggesting the activation of this biosynthetic pathway. CuGA20ox2 also had a tendency to activate 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- and non-13-hydroxylation pathways for increasing active GAs, resulting in elongated inflorescences in transgenic Arabidopsis.
Gibberellins (GAs) are plant growth substances, originally 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.) contained GA1 in its water sprouts (vegetative shoots that 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 to clarify the biosynthetic pathway of GAs in plants (for reviews: Hedden and Kamiya, 1997; Hedden and Phillips, 2000; Yamaguchi, 2008). These studies revealed that there are two pathways for synthesizing active GAs; the 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 from isopentenyl diphosphate and dimethylallyl diphosphate, and another pathway where GAs, such as GA4, are synthesized through GA15, GA24, and GA9 after the synthesis of GA12 (Fig. 1). The former and the latter pathways are known as the early-13-hydroxylation and the non-13-hydroxylation pathways, respectively, because GAs in the former pathway are hydroxylated 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 to use 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 (Goto et 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 L.) (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 that these GA 13-oxidases may be involved in the switching of the two biosynthetic pathways that diverge after GA12.
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.
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 is reported that GAs play a positive role in the flowering of Japanese cedar (Cryptomeria japonica), although the mechanism underlying the phenomenon remains to be clarified (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 seedless grapes in Japan for the first time in the world.
Production of seedless fruit is very important for breeders and growers to meet consumer demands in citriculture as well as in viticulture. Satsuma mandarin is considered absolutely seedless because it is female and male sterile in addition to parthenocarpic and very productive without pollinators (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 its precursors, such as 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 degree of 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 sweet orange (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 in clarifying the roles of these genes and the distribution of GAs in the life cycle of citrus, such as Satsuma mandarin. Satsuma mandarin originated in Japan, and is a unique and important scion cultivar providing us with seedless 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 the mechanisms of parthenocarpy and peel puffins in the future.
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 ‘Miyagawa-wase’), ‘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 for Southern 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 mandarinLeaves and ovaries of Satsuma mandarin ‘Silverhill’ were used to isolate the cDNA of GA 20-oxidase genes CuGA20ox1 and CuGA20ox2, respectively. cDNA from the 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) and CcGA20ox2(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 into a 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.
Primer sets used in gene cloning, probe labelling, quantitative real-time RT-PCR analysis, and vector construction.
Amino acid sequences were analyzed using the ClustalX multiple sequence alignment program ver. 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 the neighbor-joining (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 [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.) Bruch & Schimp [PpGA20ox (EU262757)], poplar [Populus nigra L., PnGA20ox1 (AB089675); P. Alba L., 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)], Satsuma mandarin [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 was displayed using the N-J plot unrooted (Perrière and Gouy, 1996) with bootstrap values for 1000 resamplings in each branch.
DNA extraction and hybridization analysisGenomic 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 then blotted onto Hybond-N+ nylon membranes (GE Healthcare 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 gene cloning 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 the NightOWL image analyzer (Berthold Technologies, Bad Wildbad, Germany). The primer sets used for labelling are listed in Table 1.
Expression analysis by quantitative real-time RT-PCRUsing a phenol-SDS based method as described by Ikoma et al. (1996), total RNA was extracted for analysis from seeds, roots (whole roots including lateral ones), stems, new and old leaves, and nodes of two-year-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 (in September, November, and December), and peels (in September 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, and transcript levels were normalized against each internal control.
Construction of the transformation vectorTo construct a vector for the constitutive expression of CuGA20ox1 and CuGA20ox2, the coding region of each 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 product was subsequently digested with XbaI and SacI, and then cloned 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 transformationAgrobacterium tumefaciens strain EHA101 harboring a 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 were transplanted 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 the base 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 gibberellinsTwo month after the start of incubation, whole transgenic and wild-type Arabidopsis plants were used for the quantification of 13 gibberellins on both the early-13-hydroxylation pathway (GA53, GA44, GA19, GA20, GA1, GA8, and GA29) and the non-13-hydroxylation 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 ground with 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 prepared in this experiment (n = 5, five biological replicates). After incubation for 12 h at 4°C, each homogenate was centrifuged 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 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 then loaded 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 through a 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® LC-MS/MS System (AB Sciex, Framingham, MA, USA), using an electrospray ionization (ESI) interface. The purified samples were injected onto a Shim-pack XR-ODS 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 initial condition 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 phase for 12 min before each analytical run. Quantification of the GAs was described previously (Fukuda et al., 2009).
Statistical analysisData were analyzed using a one-way ANOVA and the differences were contrasted using Tukey’s multiple comparison. All statistical analyses were perfomed at a significance level of P-value < 0.05 using R-3.2.0 (R Core Team, 2015).
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) with primer sets based on the sequences of CcGA20ox1 (AJ250187) and CcGA20ox2 (EU834066) (Huerta et al., 2009; Vidal et al., 2001). Coding sequences of Citrus unshiu GA20ox1 (CuGA20ox1; Accession No. LC056054) and Citrus unshiu GA20ox2 (CuGA20ox2; Accession No. LC056055) cDNA were 1152 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 and CcGA20ox1 and between CuGA20ox2 and CcGA20ox2 were 99.5% and 99.4%, respectively, and those at the amino acid level were 99.0% and 99.2%, respectively (Fig. 2B). In addition, at the amino acid level, 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 and the 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 cDNA of CuGA20ox1 and CuGA20ox2 showed a single band of about 10 kb for CuGA20ox1 (Fig. 3A) and polymorphic bands for CuGA20ox2 (Fig. 3B). To examine the evolutionary 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 the different 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 phylogenetically very similar or identical to those from sweet orange and Clementine as expected (Fig. 4B).
Schematic representation of the genomic organization (A) and comparison of the deduced amino acid sequence (B) of CuGA20ox1 and CuGA20ox2. 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.
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 then separated 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 followed by 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.
Phylogenetic analysis of CuGA20ox1 and CuGA20ox2 together with other GA 20-oxidases. The tree was constructed by the neighbor-joining (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)], sweet orange [CsGA20ox1 (orange1.1g016776m), CsGA20ox2 (orange1.1g041132m)], tobacco [NtGA20ox (AB012856), NsGA20ox2 (AF494088)], and tomato [LeGA20ox1 (AF049898), LeGA20ox2 (AF049899), LeGA20ox3 (AF049900), SlGA20ox4 (EU675630)].
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 adult phases using primers specific to each gene (Table 1). The transcript of CuGA20ox1 accumulated mainly in new (NL) and old (OL) leaves in both the juvenile and adult phases. It was also detected clearly in seeds and roots, 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), and juice sacs and peels in September (Fig. 5A). In contrast, the transcript of CuGA20ox2 accumulated specifically in flower buds with very low expression in peels (Fig. 5B).
Expression patterns of CuGA20ox1 (A) and CuGA20ox2 (B) in various Satsuma mandarin tissues by quantitative real-time RT-PCR. The samples for (A) and (B) from left to right are 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 in this experiment and the PCR conditions are described in Table 1.
To elucidate the function of GA 20-oxidase-like genes of Satsuma mandarin, CuGA20ox1 and CuGA20ox2, we introduced the coding region of each gene 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 of days 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 to flowering, 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): wild-type plants; Vector/wt (Col): transgenic lines with an empty vector]. However, transgenic lines 1-1, 1-2, and 1-5 of 35SΩ::CuGA20ox1 with values of 23.19, 23.00, and 22.81 cm, respectively, and transgenic lines 2-1 and 2-3 of 35SΩ::CuGA20ox2 with values of 23.96 and 23.46 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).
Schematic representation of transformation vectors (A) and typical 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 an empty vector.
Characteristics of transgenic lines ectopically expressing CuGA20ox1 or CuGA20ox2.
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 the highest 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 in decreasing order (Fig. 7B). As expected, the lines with relatively higher expression of each transgene, such as lines 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).
Expression analysis for transgenes in whole plants of transgenic Arabidopsis. (A) Gene expression of CuGA20ox1 in transgenic lines. (B) Gene expression of CuGA20ox2 in transgenic lines. Levels of detected amplicons were normalized by reference to CuActin. Values are means ± SD of the results from three replicates per line. Asterisks represent the lines that exhibit an elongated phenotype with a statistically significant difference from the wild-type controls shown in Table 2.
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 a wild-type control (Fig. 8B). On the non-13-hydroxylation pathway in line 1, GA24 decreased, whereas GA34 increased significantly (Fig. 8E, H). In transgenic lines with 35SΩ::CuGA20ox2, designated as line 2, GA20 increased significantly on the early-13-hydroxylation pathway (Fig. 8B). For other 13-OH GAs in line 2, changes in the levels of these GAs were not significantly 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).
Evaluation of eight GAs on both the early-13-hydroxylation and the non-13-hydroxylation pathways in transgenic Arabidopsis. (A–D) Concentrations of GA19, GA20, GA24, and GA8 on the early-13-hydroxylation pathway. (E–H) Concentrations of GA24, GA9, GA51, and GA34 on the non-13-hydroxylation pathway. Ten samples (one sample per each independent line of 35S::CuGA20ox1 or 35S::CuGA20ox2) were prepared and subjected to LC-MS/MS measurement in this experiment (n = 5, five biological replicates). Line 1 and line 2 represent transgenic lines with 35S::CuGA20ox1 and 35S::CuGA20ox2, respectively. Values are means ± SD. One or two asterisks indicate a statistically significant difference from the wild-type plants in the same column (P < 0.05, 0.01, respectively). ND, not detected.
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 that in Clementine and very similar to that in Carrizo citrange and sweet orange; the amino acid sequence deduced 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 trifoliate orange have two types of GA 20-oxidase genes. Especially, the blot probed with CuGA20ox2 showed polymorphic bands, indicating the diversity of citrus GA20ox2 genes, although the blot probed with CuGA20ox1 showed single bands at a similar molecular weight, indicating conserved sequences around the XbaI site in citrus GA20ox1 genes. In considering 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 that is also seen in a pummelo cultivar (‘Benimadoka’). The deduced amino acid sequences of CuGA20ox1 and CuGA20ox2 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 the function of these two genes diverged in the process of evolution. 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 in flower buds would explain why GA-like activity is higher 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 around anthesis (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 and CuGA20ox2 caused elongated inflorescence, but did not affect 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, the lines with relatively higher expression of CuGA20ox1 or CuGA20ox2 showed an elongated phenotype with a statistically 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 than those 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 the effect of these gene products on the GA biosynthetic pathways in transgenic Arabidopsis. Consistent with the fact that Arabidopsis mainly utilizes the non-13-hydroxylation pathway to accumulate active GAs such as GA4, the concentrations of GA9, GA51, and GA34 (Fig. 8F–H) are relatively higher on the non-13-hydroxylation pathway than are concentrations of GA20, GA29, and GA8 (Fig. 8B–D), respectively, on the early-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 were entering 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 and GA34 increased (Fig. 8E, H). This observation indicated that both CuGA20ox1 and CuGA20ox2 accelerated the convertion of GA24, a substrate of a GA 20-oxidase, to GA9, a precursor of an active form of GA4. In addition, an increased concentration of GA34 suggested that most active 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 and increased 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 less GA1 and more GA8 than those of wild type plants and also suggeted that the content of GA8 may be a measure of 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 the early-13-hydroxylation pathway although it increased only GA20 with a statistically significant difference (Fig. 8B). Taken together, we concluded that CuGA20ox1 and/or CuGA20ox2 activate both the early-13- and non-13-hydroxylation pathways, resulting in elongated inflorescences in transgenic Arabidopsis. CcGA20ox1 was confirmed to encode for a GA 20-oxidase 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 and coordinate the GA biosynthetic pathways in citrus must be studied to clarify more than a few physiological phenomena, such as flowering, fruit development and seedlessness, mediated by GA signal transduction.
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 Y. Uchida for their technical assistance.
