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. 1999 Oct;121(2):373-82.
doi: 10.1104/pp.121.2.373.

Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon

H G Kang  1 , S H Jun, J Kim, H Kawaide, Y Kamiya, G An
Affiliations

Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon

H G Kang et al. Plant Physiol. 1999 Oct.

Abstract

To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA(12) at C-20 to the C(19) compound GA(9), a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the beta-glucuronidase (GUS) gene. In a transient expression system, beta-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds.

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Figures

Figure 1
Figure 1
Alignment of the deduced amino acid sequence for the Cv20ox cDNA with GA 20-oxidases from different species. Regions identical to all GA 20-oxidases are boxed in black. The number symbols (#) indicates the putative Fe2+-binding motif typical for 2-oxoglutarate-dependent dioxygenases, and the asterisks (*) indicate the putative 2-oxoglutarate-binding motif. Dashes were introduced for the maximum sequence homology.
Figure 2
Figure 2
Genomic DNA-blot analysis of Cv20ox. Ten micrograms of watermelon genomic DNA was digested with EcoRI (E) or HindIII (H) and resolved on a 0.8% agarose gel. The 300-bp cDNA cloned by PCR was used as a probe. The positions of HindIII-digested λ-DNA size markers are shown on the right in kb.
Figure 3
Figure 3
Spatial and temporal expression of Cv20ox. A, The blot of the RNAs isolated from different parts of the mature plant was hybridized with the same probe as that used in genomic DNA-blot analysis. I, Integuments (8 DAP); S, whole seeds (8 DAP); IS, inner seed tissues (8 DAP); F1, fruits (1 DAP); F2, fruits (2–3 DAP); F8, fruits (8 DAP); T, tendrils; L, leaves; M, male flowers; O, ovaries before pollination. B, RNAs isolated from different organs of young plants grown for 15 d after imbibition were blotted and hybridized with the same probe as that described above. C, Cotyledons; Sm, shoot meristem including uppermost of hypocotyls; Hu, upper hypocotyls; Hl, lower hypocotyls; R, roots. Twenty-five micrograms of RNA was used for the blot analyses. C, Temporal expression pattern of Cv20ox during seed development. Twenty micrograms of RNAs isolated from whole seeds at seven different stages after pollination was resolved on a 0.8% agarose gel, transferred onto a nylon membrane, and hybridized with the Cv20ox probe.
Figure 4
Figure 4
Effects of GA3 treatment on Cv20ox gene expression. +, 3 DAP fruits injected with about 300 μL of 2 × 10−5 m GA3 dissolved in 0.1% Tween 20, and seeds were harvested 24 h after the treatment. −, Control RNA taken from seeds of fruits injected with 0.1% Tween 20 solution. The same blot was washed and rehybridized with the Suc synthase cDNA probe to confirm equal loading of RNAs.
Figure 5
Figure 5
Localization of the Cv20ox mRNA in watermelon seeds by in situ hybridization. All samples were sectioned longitudinally in parallel with the plane of seed. Micrographs show pollinated seeds at 5 DAP (A, B, and C), at 6 DAP (D and E), and at 4 DAP (F and G). The sections were hybridized to either an antisense (B, E, and G) or a sense (C) RNA probe of the entire cDNA that was labeled with digoxigenin. A, D, and F are toluidine-blue-stained samples. nu, Nucellus; ii, inner layer of integument; oi, outer layers of integument; mp, micropylar end; e, globular embryo. Bar = 100 μm.
Figure 6
Figure 6
Effects of CPPU on Cv20ox gene expression. A, Comparative RNA-blot analysis of the Cv20ox gene between seeds harvested after normal pollination and after CPPU treatment. Lanes 1, 2, and 3, Seeds at 2, 3, and 4 DAP, respectively. Lanes 4, 5, and 6 are samples from 2, 3, and 4 d after CPPU treatment, respectively. B, The Cv20ox mRNA expression pattern in integuments (1) and inner seed tissues (2) of seeds at 10 d after CPPU treatment.
Figure 7
Figure 7
A, Nucleotide sequence of the 5′-upstream region of the Cv20ox gene. The putative TATA box is double-underlined and the translation start codon is in bold. Sequences consisting of 19 bp or more A/T are underlined. B, Plasmid map of pGA2118. The terminator region of the nopaline synthase gene (nosT) was placed after the GUS coding region. H, HindIII; Sp, SpeI; N, NotI; Bs, BstZI; Sa, SalI; Xb, XbaI; B, BamHI; Sm, SmaI; P, PstI; S, SacI; E, EcoRI; K, KpnI; X, XhoI; St, StuI.
Figure 8
Figure 8
Histochemical detection of GUS expression in different organs of watermelon plants after bombardment with tungsten particles coated with pGA2118. A, Developing seeds (8 DAP); B, inside of the seeds cut in parallel with the plane of the seeds (8 DAP); C to F, leaves, roots, hypocotyl, and cotyledons of seedlings, respectively.
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